U.S. patent application number 10/285976 was filed with the patent office on 2003-09-04 for wnt and frizzled receptors as targets for immunotherapy in head and neck squamous cell carcinomas.
This patent application is currently assigned to REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Carson, Dennis A., Corr, Maripat, Leoni, Lorenzo M., Rhee, Chae-Seo, Sen, Malini, Wu, Christina.
Application Number | 20030165500 10/285976 |
Document ID | / |
Family ID | 32312059 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030165500 |
Kind Code |
A1 |
Rhee, Chae-Seo ; et
al. |
September 4, 2003 |
Wnt and frizzled receptors as targets for immunotherapy in head and
neck squamous cell carcinomas
Abstract
The diverse receptor-ligand pairs of the Wnt and frizzled (Fzd)
families play important roles during embryonic development, and
thus may be overexpressed in cancers that arise from immature
cells. The mRNA levels and expression levels of 5 Wnt (Wnt-1, 5a,
7a, 10b, 13) and 2 Fzd (Fzd-2, 5) genes in 10 head and neck
squamous carcinoma cell lines (HNSCC) were investigated. In
addition, anti-Wnt-1 antibodies were used to study the Wnt/Fzd
signalling pathway. These results indicate that HNSCC cell lines
overexpress one or more Wnt and Fzd genes, and the proliferation
and survival of a subset of HNSCC may depend on the Wnt/Fzd
pathway. Therefore, the Wnt and Fzd receptors may be useful targets
for immunotherapy of this common cancer.
Inventors: |
Rhee, Chae-Seo; (Seoul,
KR) ; Sen, Malini; (San Diego, CA) ; Wu,
Christina; (San Diego, CA) ; Leoni, Lorenzo M.;
(San Diego, CA) ; Corr, Maripat; (San Diego,
CA) ; Carson, Dennis A.; (Del Mar, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
REGENTS OF THE UNIVERSITY OF
CALIFORNIA
Oakland
CA
|
Family ID: |
32312059 |
Appl. No.: |
10/285976 |
Filed: |
November 1, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10285976 |
Nov 1, 2002 |
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PCT/US02/13802 |
May 1, 2002 |
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60287995 |
May 1, 2001 |
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Current U.S.
Class: |
424/143.1 |
Current CPC
Class: |
C12Q 1/6851 20130101;
C12Q 2600/158 20130101; C12Q 1/6886 20130101; A61P 35/00 20180101;
C07K 16/3015 20130101; A61P 35/02 20180101; C07K 2317/73 20130101;
C07K 16/3023 20130101; C07K 16/3061 20130101; C07K 16/22 20130101;
C07K 16/2863 20130101; A61K 2039/505 20130101 |
Class at
Publication: |
424/143.1 |
International
Class: |
A61K 039/395 |
Goverment Interests
[0002] This invention was made with U.S. Government support under
Grant AR 44850 awarded by the National Institutes of Health. The
Government may have certain rights in this invention.
Claims
What is claimed is:
1. A method of inhibiting the proliferation or survival of breast
cancer cells, wherein the cancer cells overexpress a Wnt protein in
a Wnt/Fzd signaling pathway when compared to non-cancer cells, and
wherein the Wnt protein is selected from the group consisting of
Wnt7b, Wnt-10b, and Wnt-14, said method comprising contacting the
cancer cells with an agent that inhibits the Wnt/Fzd signaling
pathway in the cancer cells.
2. The method according to claim 1, wherein the agent is an
antagonist of the Wnt/Fzd signaling pathway.
3. The method according to claim 2, wherein the agent is an
anti-Wnt antibody that specifically binds Wnt7b, Wnt-10b, or
Wnt-14.
4. The method according to claim 3, wherein the anti-Wnt antibody
facilitates cellular toxicity or killing by complement.
5. The method according to claim 1, wherein the Wnt protein is
overexpressed when compared to another Wnt protein in the same
cancer cells.
6. The method according to claim 1, wherein the Wnt protein is
required for proliferation or survival of the cancer cell.
7. A method of treating a patient with a breast cancer, wherein the
cancer cells overexpress a Wnt protein in a Wnt/Fzd signaling
pathway when compared to non-cancer cells, and wherein the Wnt
protein is selected from the group consisting of Wnt7b, Wnt8a, and
Wnt-14, said method comprising contacting the cancer cells with an
agent that inhibits the Wnt/Fzd signaling pathway in the cancer
cells.
8. The method according to claim 7, wherein the agent is an
antagonist of the Wnt/Fzd signaling pathway.
9. The method according to claim 8, wherein the agent is an
anti-Wnt antibody that specifically binds Wnt7b, Wnt-10b, or
Wnt-14.
10. The method according to claim 9, wherein the anti-Wnt antibody
facilitates cellular toxicity or killing by complement.
11. The method according to claim 7, wherein the Wnt protein is
overexpressed when compared to another Wnt protein in the same
cancer cells.
12. The method according to claim 7, wherein the Wnt protein is
required for proliferation or survival of the cancer cell.
13. A method of inhibiting the proliferation or survival of chronic
lymphocytic leukemia cells, wherein the cancer cells overexpress a
Wnt protein in a Wnt/Fzd signaling pathway when compared to
non-cancer cells, and wherein the Wnt protein is selected from the
group consisting of Wnt3 and Wnt-16, said method comprising
contacting the cancer cells with an agent that inhibits the Wnt/Fzd
signaling pathway in the cancer cells.
14. The method according to claim 13, wherein the agent is an
antagonist of the Wnt/Fzd signaling pathway.
15. The method according to claim 14, wherein the agent is an
anti-Wnt antibody that specifically binds Wnt3, or Wnt-16.
16. The method according to claim 15, wherein the anti-Wnt antibody
facilitates cellular toxicity or killing by complement.
17. The method according to claim 13, wherein the Wnt protein is
overexpressed when compared to another Wnt protein in the same
cancer cells.
18. The method according to claim 13, wherein the Wnt protein is
required for proliferation or survival of the cancer cell.
19. A method of treating a patient with chronic lymphocytic
leukemia, wherein the cancer cells overexpress a Wnt protein in a
Wnt/Fzd signaling pathway when compared to non-cancer cells, and
wherein the Wnt protein is selected from the group consisting of
Wnt3 and Wnt-16, said method comprising contacting the cancer cells
with an agent that inhibits the Wnt/Fzd signaling pathway in the
cancer cells.
20. The method according to claim 19, wherein the agent is an
antagonist of the Wnt/Fzd signaling pathway.
21. The method according to claim 20, wherein the agent is an
anti-Wnt antibody that specifically binds Wnt3, or Wnt-16.
22. The method according to claim 21, wherein the anti-Wnt antibody
facilitates cellular toxicity or killing by complement.
23. The method according to claim 19, wherein the Wnt protein is
overexpressed when compared to another Wnt protein in the same
cancer cells.
24. The method according to claim 19, wherein the Wnt protein is
required for proliferation or survival of the cancer cell.
25. A method of inhibiting the proliferation or survival of mantle
zone lymphoma cells, wherein the cancer cells overexpress a Wnt
protein in a Wnt/Fzd signaling pathway when compared to non-cancer
cells, and wherein the Wnt protein is Wnt-16, said method
comprising contacting the cancer cells with an agent that inhibits
the Wnt/Fzd signaling pathway in the cancer cells.
26. The method according to claim 25, wherein the agent is an
antagonist of the Wnt/Fzd signaling pathway.
27. The method according to claim 26, wherein the agent is an
anti-Wnt antibody that specifically binds Wnt-16.
28. The method according to claim 27, wherein the anti-Wnt antibody
facilitates cellular toxicity or killing by complement.
29. The method according to claim 25, wherein the Wnt protein is
overexpressed when compared to another Wnt protein in the same
cancer cells.
30. The method according to claim 25, wherein the Wnt protein is
required for proliferation or survival of the cancer cell.
31. A method of treating a patient with mantle zone lymphoma,
wherein the cancer cells overexpress a Wnt protein in a Wnt/Fzd
signaling pathway when compared to non-cancer cells, and wherein
the Wnt protein is Wnt-16, said method comprising contacting the
cancer cells with an agent that inhibits the Wnt/Fzd signaling
pathway in the cancer cells.
32. The method according to claim 31, wherein the agent is an
antagonist of the Wnt/Fzd signaling pathway.
33. The method according to claim 32, wherein the agent is an
anti-Wnt antibody that specifically binds Wnt-16.
34. The method according to claim 33 wherein the anti-Wnt antibody
facilitates cellular toxicity or killing by complement.
35. The method according to claim 31, wherein the Wnt protein is
overexpressed when compared to another Wnt protein in the same
cancer cells.
36. The method according to claim 31, wherein the Wnt protein is
required for proliferation or survival of the cancer cell.
37. A method of inhibiting the proliferation or survival of breast
cancer cells, wherein the cancer cells overexpress a Fzd protein in
a Wnt/Fzd signaling pathway when compared to non-cancer cells, and
wherein the Fzd protein is selected from the group consisting of
Fzd3, Fzd4, Fzd6, Fzd7, or Fzd10, said method comprising contacting
the cancer cells with an agent that inhibits the Wnt/Fzd signaling
pathway in the cancer cells.
38. The method according to claim 37, wherein the agent is an
antagonist of the Wnt/Fzd signaling pathway.
39. The method according to claim 38, wherein the agent is an
anti-Fzd antibody that specifically binds Fzd3, Fzd4, Fzd6, Fzd7,
or Fzd10.
40. The method according to claim 39, wherein the anti-Fzd antibody
facilitates cellular toxicity or killing by complement.
41. The method according to claim 37, wherein the Fzd protein is
overexpressed when compared to another Fzd protein in the same
cancer cells.
42. The method according to claim 37, wherein the Fzd protein is
required for proliferation or survival of the cancer cell.
43. A method of treating a patient with a breast cancer, wherein
the cancer cells overexpress the Wnt protein when compared to
non-cancer cells, and wherein the Fzd protein is selected from the
group consisting of Fzd3, Fzd4, Fzd6, Fzd7, or Fzd10, said method
comprising administering to the patient an agent that inhibits the
Wnt/Fzd signaling pathway in the cancer cells.
44. The method according to claim 43, wherein the agent is an
antagonist of the Wnt/Fzd signaling pathway.
45. The method according to claim 44, wherein the agent is an
anti-Fzd antibody that specifically binds Fzd3, Fzd4, Fzd6, Fzd7,
or Fzd10.
46. The method according to claim 45, wherein the anti-Fzd antibody
facilitates cellular toxicity or killing by complement.
47. The method according to claim 43, wherein the Fzd protein is
overexpressed when compared to another Fzd protein in the same
cancer cells.
48. The method according to claim 43, wherein the Fzd protein is
required for proliferation or survival of the cancer cell.
49. A method of inhibiting the proliferation or survival of chronic
lymphocytic leukemia cells, wherein the cancer cells overexpress a
Fzd protein in a Wnt/Fzd signaling pathway when compared to
non-cancer cells, and wherein the Fzd protein is Fzd3, said method
comprising contacting the cancer cells with an agent that inhibits
the Wnt/Fzd signaling pathway in the cancer cells.
50. The method according to claim 49, wherein the agent is an
antagonist of the Wnt/Fzd signaling pathway.
51. The method according to claim 50, wherein the agent is an
anti-Fzd antibody that specifically binds Fzd3.
52. The method according to claim 51, wherein the anti-Fzd antibody
facilitates cellular toxicity or killing by complement.
53. The method according to claim 49, wherein the Fzd protein is
overexpressed when compared to another Fzd protein in the same
cancer cells.
54. The method according to claim 49, wherein the Fzd protein is
required for proliferation or survival of the cancer cell.
55. A method of treating a patient with chronic lymphocytic
leukemia, wherein the cancer cells overexpress a Fzd protein in a
Wnt/Fzd signaling pathway when compared to non-cancer cells, and
wherein the Fzd protein is Fzd3, said method comprising contacting
the cancer cells with an agent that inhibits the Wnt/Fzd signaling
pathway in the cancer cells.
56. The method according to claim 55, wherein the agent is an
antagonist of the Wnt/Fzd signaling pathway.
57. The method according to claim 56, wherein the agent is an
anti-Fzd antibody that specifically binds Fzd3.
58. The method according to claim 57, wherein the anti-Fzd antibody
facilitates cellular toxicity or killing by complement.
59. The method according to claim 55, wherein the Fzd protein is
overexpressed when compared to another Fzd protein in the same
cancer cells.
60. The method according to claim 55, wherein the Fzd protein is
required for proliferation or survival of the cancer cell.
61. A method of inhibiting the proliferation or survival of cancer
cells, wherein the cancer cells overexpress a Wnt protein when
compared to non-cancer cells, and wherein the cancer cells
overexpress a downstream wnt/fzd regulated gene product compared to
non-cancer cells, said method comprising contacting the cancer
cells with an agent that inhibits the Wnt/Fzd signaling pathway in
the cancer cells.
62. The method according to claim 61, wherein the Wnt protein is
overexpressed when compared to another Wnt protein in the same
cancer cells.
63. The method according to claim 61, wherein the Wnt protein is
required for proliferation or survival of the cancer cell.
64. The method according to claim 61, wherein the cancer is breast
cancer.
65. The method according to claim 61, wherein the Wnt protein is
selected from the group consisting of Wnt7b, Wnt-10b, and
Wnt-14.
66. The method according to claim 61, wherein the downstream
wnt/fzd regulated gene product is selected from the group
consisting of cyclin D1, c-myc, and WISP2.
67. A method of inhibiting the proliferation or survival of cancer
cells, wherein the cancer cells overexpress a Fzd protein when
compared to non-cancer cells, and wherein the cancer cells
overexpress a downstream wnt/fzd regulated gene product, said
method comprising contacting the cancer cells with an agent that
inhibits the Wnt/Fzd signaling pathway in the cancer cells.
68. The method according to claim 67, wherein the Fzd protein is
overexpressed when compared to another Fzd protein in the same
cancer cells.
69. The method according to claim 67, wherein the Fzd protein is
required for proliferation or survival of the cancer cell.
70. The method according to claim 67, wherein the cancer is breast
cancer.
71. The method according to claim 67, wherein the Fzd protein is
selected from the group consisting of Fzd3, Fzd4, Fzd6, Fzd7, and
Fzd10.
72. The method according to claim 67, wherein the downstream
wnt/fzd regulated gene product is selected from the group
consisting of cyclin D1, c-myc, and WISP2.
73. A method of treating a patient with a cancer, wherein the
cancer cells overexpress a Wnt protein when compared to non-cancer
cells, and wherein the cancer cells overexpress a downstream
wnt/fzd regulated gene product compared to non-cancer cells, said
method comprising administering to the patient an agent that
inhibits the Wnt/Fzd signaling pathway in the cancer cells.
74. The method according to claim 73, wherein the Wnt protein is
overexpressed when compared to another Wnt protein in the same
cancer cells.
75. The method according to claim 73, wherein the Wnt protein is
required for proliferation or survival of the cancer cell.
76. The method according to claim 73, wherein the cancer is breast
cancer.
77. The method according to claim 73, wherein the Wnt protein is
selected from the group consisting of Wnt7b, Wnt-10b, and
Wnt-14.
78. The method according to claim 73, wherein the downstream
wnt/fzd regulated gene product is selected from the group
consisting of cyclin D1, c-myc, and WISP2.
79. A method of treating a patient with a cancer, wherein the
cancer cells overexpress a Fzd protein when compared to non-cancer
cells, and wherein the cancer cells overexpress a downstream
wnt/fzd regulated gene product compared to non-cancer cells, said
method comprising administering to the patient an agent that
inhibits the Wnt/Fzd signaling pathway in the cancer cells.
80. The method according to claim 79, wherein the Fzd protein is
overexpressed when compared to another Fzd protein in the same
cancer cells.
81. The method according to claim 79, wherein the Fzd protein is
required for proliferation or survival of the cancer cell.
82. The method according to claim 79, wherein the cancer is breast
cancer.
83. The method according to claim 79, wherein the Fzd protein is
selected from the group consisting of Fzd3, Fzd4, Fzd6, Fzd7, and
Fzd10.
84. The method according to claim 79, wherein the downstream
wnt/fzd regulated gene product is selected from the group
consisting of cyclin D1, c-myc, and WISP2.
85. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Wnt-1 encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-CGAACCTGCTTACAGACTCCAA-3' and
5'-CAGACGCCGCTGTTTGC-3'.
86. A method of specifically detecting the presence or absence of a
nucleic acid encoding Wnt-1 in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim 85,
under conditions to permit specific hybridization between Wnt-l
encoding nucleic acid present in the sample and the polynucleotide
and detecting whether the specific hybridization occurs.
87. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Wnt-2 encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-GGATGACCAAGTGTGGGTGTAAG-3' and
5'-GTGCACATCCAGAGCTTCCA-3'.
88. A method of specifically detecting the presence or absence of a
nucleic acid encoding Wnt-2 in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim 87,
under conditions to permit specific hybridization between Wnt-2
encoding nucleic acid present in the sample and the polynucleotide
and detecting whether the specific hybridization occurs.
89. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Wnt-2b encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-GGCACGAGTGATCTGTGACAATA-3' and
5'-CGCATGATGTCTGGGTAACG-3'.
90. A method of specifically detecting the presence or absence of a
nucleic acid encoding Wnt-2b in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim 87,
under conditions to permit specific hybridization between Wnt-2b
encoding nucleic acid present in the sample and the polynucleotide
and detecting whether the specific hybridization occurs.
91. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Wnt-3 encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-CTGGGCCAGCAGTACACATCT-3' and
5'-GGCATGATCTCGATGTAATTGC-3'.
92. A method of specifically detecting the presence or absence of a
nucleic acid encoding Wnt-3 in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim 91,
under conditions to permit specific hybridization between Wnt-3
encoding nucleic acid present in the sample and the polynucleotide
and detecting whether the specific hybridization occurs.
93. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Wnt-3a encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-CCCGTGCTGGACAAAGCT-3' and
5'-TCTGCACATGAGCGTGTCACT-3'.
94. A method of specifically detecting the presence or absence of a
nucleic acid encoding Wnt-3a in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim 93,
under conditions to permit specific hybridization between Wnt-3a
encoding nucleic acid present in the sample and the polynucleotide
and detecting whether the specific hybridization occurs.
95. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Wnt-4 encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-GGAGGAGACGTGCGAGAAAC-3' and
5'-CAGGTTCCGCTTGCACATCT-3'.
96. A method of specifically detecting the presence or absence of a
nucleic acid encoding Wnt-4 in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim 95,
under conditions to permit specific hybridization between Wnt-4
encoding nucleic acid present in the sample and the polynucleotide
and detecting whether the specific hybridization occurs.
97. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Wnt-5a encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-TCTCCTTCGCCCAGGTTGTA-3' and
5'-CTTCTGACATCTGAACAGGGTTATTC-3'.
98. A method of specifically detecting the presence or absence of a
nucleic acid encoding Wnt-5a in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim 97,
under conditions to permit specific hybridization between Wnt-5a
encoding nucleic acid present in the sample and the polynucleotide
and detecting whether the specific hybridization occurs.
99. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Wnt-5b encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-CCAACTCCTGGTGGTCATTAGC-3' and
5'-TGGGCACCGATGATAAACATC-3'.
100. A method of specifically detecting the presence or absence of
a nucleic acid encoding Wnt-5b in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim 99,
under conditions to permit specific hybridization between Wnt-5b
encoding nucleic acid present in the sample and the polynucleotide
and detecting whether the specific hybridization occurs.
101. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Wnt-6 encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-TCCGCCGCTGGAATTG-3' and
5'-AGGCCGTCTCCCGAATGT-3'.
102. A method of specifically detecting the presence or absence of
a nucleic acid encoding Wnt-6 in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
101, under conditions to permit specific hybridization between
Wnt-6 encoding nucleic acid present in the sample and the
polynucleotide and detecting whether the specific hybridization
occurs.
103. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Wnt-7a encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-GACGCCATCATCGTCATAGGA-3' and
5'-GGCCATTGCGGAACTGAA-3'.
104. A method of specifically detecting the presence or absence of
a nucleic acid encoding Wnt-7a in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
103, under conditions to permit specific hybridization between
Wnt-7a encoding nucleic acid present in the sample and the
polynucleotide and detecting whether the specific hybridization
occurs.
105. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Wnt-7b encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-TGAAGCTCGGAGCACTGTCA-3' and
5'-GGCCAGGAATCTTGTTGCA-3'.
106. A method of specifically detecting the presence or absence of
a nucleic acid encoding Wnt-7b in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
105, under conditions to permit specific hybridization between
Wnt-7b encoding nucleic acid present in the sample and the
polynucleotide and detecting whether the specific hybridization
occurs.
107. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Wnt-8a encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-GCAGAGGCGGAACTGATCTT-3' and
5'-CGACCCTCTGTGCCATAGATG-3'.
108. A method of specifically detecting the presence or absence of
a nucleic acid encoding Wnt-8a in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
107, under conditions to permit specific hybridization between
Wnt-8a encoding nucleic acid present in the sample and the
polynucleotide and detecting whether the specific hybridization
occurs.
109. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Wnt-8b encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-AATCGGGAGACAGCATTTGTG-3' and
5'-ATCTCCAAGGCTGCAGTTTCTAGT-3'.
110. A method of specifically detecting the presence or absence of
a nucleic acid encoding Wnt-8b in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
109, under conditions to permit specific hybridization between
Wnt-8b encoding nucleic acid present in the sample and the
polynucleotide and detecting whether the specific hybridization
occurs.
111. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Wnt-10a encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-CTGGGTGCTCCTGTTCTTCCTA-3' and
5'-GAGGCGGAGGTCCAGAATG-3'.
112. A method of specifically detecting the presence or absence of
a nucleic acid encoding Wnt-10a in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
111, under conditions to permit specific hybridization between
Wnt-10a encoding nucleic acid present in the sample and the
polynucleotide and detecting whether the specific hybridization
occurs.
113. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Wnt-10b encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-CCTCGCGGGTCTCCTGTT-3' and
5'-AGGCCCAGAATCTCATTGCTTA-3'.
114. A method of specifically detecting the presence or absence of
a nucleic acid encoding Wnt-10b in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
113, under conditions to permit specific hybridization between
Wnt-10b encoding nucleic acid present in the sample and the
polynucleotide and detecting whether the specific hybridization
occurs.
115. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Wnt-1 encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-CGTGTGCTATGGCATCAAGTG-3' and
5'-GCAGTGTTGCGTCTGGTTCA-3'.
116. A method of specifically detecting the presence or absence of
a nucleic acid encoding Wnt-11 in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
115, under conditions to permit specific hybridization between
Wnt-11 encoding nucleic acid present in the sample and the
polynucleotide and detecting whether the specific hybridization
occurs.
117. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Wnt-14 encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-GGGCAGACGGTCAAGCAA-3' and
5'-CCAGCCTTGATCACCTTCACA-3'.
118. A method of specifically detecting the presence or absence of
a nucleic acid encoding Wnt-14 in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
117, under conditions to permit specific hybridization between
Wnt-14 encoding nucleic acid present in the sample and the
polynucleotide and detecting whether the specific hybridization
occurs.
119. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Wnt-16 encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-GCCAATTTGCCGCTGAAC-3' and
5'-CGGCAGCAGGTACGGTTT-3'.
120. A method of specifically detecting the presence or absence of
a nucleic acid encoding Wnt-16 in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
119, under conditions to permit specific hybridization between
Wnt-16 encoding nucleic acid present in the sample and the
polynucleotide and detecting whether the specific hybridization
occurs.
121. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Fzd1 encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-CACCTTGTGAGCCGACCAA-3' and
5'-CAGCACTGACCAAATGCCAAT-3'.
122. A method of specifically detecting the presence or absence of
a nucleic acid encoding Fzd1 in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
121, under conditions to permit specific hybridization between Fzd1
encoding nucleic acid present in the sample and the polynucleotide
and detecting whether the specific hybridization occurs.
123. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Fzd2 encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-TTTCTGGGCGAGCGTGAT-3' and
5'-AAACGCGTCTCCTCCTGTGA-3'.
124. A method of specifically detecting the presence or absence of
a nucleic acid encoding Fzd2 in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
123, under conditions to permit specific hybridization between Fzd2
encoding nucleic acid present in the sample and the polynucleotide
and detecting whether the specific hybridization occurs.
125. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Fzd3 encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-TGGCTATGGTGGATGATCAAAG-3' and
5'-TGGAGGCTGCCGTGGTA-3'.
126. A method of specifically detecting the presence or absence of
a nucleic acid encoding Fzd3 in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
125, under conditions to permit specific hybridization between Fzd3
encoding nucleic acid present in the sample and the polynucleotide
and detecting whether the specific hybridization occurs.
127. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Fzd4 encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-GGCGGCATGTGTCTTTCAGT-3' and
5'-GAATTTGCTGCAGTTCAGACTCTCT-3'.
128. A method of specifically detecting the presence or absence of
a nucleic acid encoding Fzd4 in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
127, under conditions to permit specific hybridization between Fzd4
encoding nucleic acid present in the sample and the polynucleotide
and detecting whether the specific hybridization occurs.
129. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Fzd5 encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-CGCGAGCACAACCACATC-3' and
5'-AGAAGTAGACCAGGAGGAAGACGAT-3'.
130. A method of specifically detecting the presence or absence of
a nucleic acid encoding Fzd5 in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
129, under conditions to permit specific hybridization between Fzd5
encoding nucleic acid present in the sample and the polynucleotide
and detecting whether the specific hybridization occurs.
131. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Fzd6 encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-ACAAGCTGAAGGTCATTTCCAAA-3' and
5'-GCTACTGCAGAAGTGCCATGAT-3'.
132. A method of specifically detecting the presence or absence of
a nucleic acid encoding Fzd6 in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
131, under conditions to permit specific hybridization between Fzd6
encoding nucleic acid present in the sample and the polynucleotide
and detecting whether the specific hybridization occurs.
133. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Fzd7 encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-CAACGGCCTGATGTACTTTAAGG-3' and
5'-CATGTCCACCAGGTAGGTGAGA-3'.
134. A method of specifically detecting the presence or absence of
a nucleic acid encoding Fzd7 in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
133, under conditions to permit specific hybridization between Fzd7
encoding nucleic acid present in the sample and the polynucleotide
and detecting whether the specific hybridization occurs.
135. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Fzd8 encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-GCTCGGTCATCAAGCAACAG-3' and
5'-ACGGTGTAGAGCACGGTGAAC-3'.
136. A method of specifically detecting the presence or absence of
a nucleic acid encoding Fzd8 in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
135, under conditions to permit specific hybridization between Fzd8
encoding nucleic acid present in the sample and the polynucleotide
and detecting whether the specific hybridization occurs.
137. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Fzd9 encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-GCGCTCAAGACCATCGTCAT-3' and
5'-ATCCGTGCTGGCCACGTA-3'.
138. A method of specifically detecting the presence or absence of
a nucleic acid encoding Fzd9 in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
137, under conditions to permit specific hybridization between Fzd9
encoding nucleic acid present in the sample and the polynucleotide
and detecting whether the specific hybridization occurs.
139. An isolated polynucleotide of less than about 100 nucleotides
that specifically hybridizes to a Fzd10 encoding nucleic acid,
wherein the isolated polynucleotide specifically hybridizes to the
same sequence as a polynucleotide selected from the group
consisting of 5'-GCCGCCATCAGCTCCAT-3' and
5'-TCATGTTGTAGCCGATGTCCTT-3'.
140. A method of specifically detecting the presence or absence of
a nucleic acid encoding Fzd10 in a biological sample, the method
comprising contacting the sample with a polynucleotide of claim
139, under conditions to permit specific hybridization between
Fzd10 encoding nucleic acid present in the sample and the
polynucleotide and detecting whether the specific hybridization
occurs.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This patent application claims the benefit of the filing
date of U.S. Provisional Application No. 60/287,995, filed May 1,
2001 and PCT US02/13802, filed May 1, 2002, both of which are
incorporated herein by reference. Related applications U.S. Ser.
No. 09/847,102 filed May 1, 2001 and PCT/IB02/02887 filed May 1,
2002 are herein incorporated by reference.
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED ON A COMPACT DISK.
[0003] This application relates to proteins involved in the
Wnt/frizzled signaling pathway. More specifically, it involves the
role of these proteins in proliferative disorders.
BACKGROUND OF THE INVENTION
[0004] Many cancers arise from differentiated tissues that are
slowly dividing. The initial malignant population may have
developed from a small, rapidly proliferating population of
residual tissue stem cells or cells with a less differentiated
subcellular profile. A strategy for targeting tumor cells that are
antigenically distinct from mature differentiated cells could be
useful in the treatment of cancer, particularly for controlling
microscopic spread of disease. Malignant cells may express
receptors used in embryonic patterning, which may serve as
immunologic targets distinct from mature differentiated tissue.
[0005] In embryogenesis body patterning is related to the axial
expression of different proteins. The proximal-distal axis is
controlled by fibroblast growth factor (Vogel, A. et al.,
"Involvement of FGF-8 in initiation, outgrowth and patterning of
the vertebrate limb," Development, 122:1737-1750 (1996); Vogel, A.
and Tickle, C., "FGF-4 maintains polarizing activity of posterior
limb bud cells in vivo and in vitro," Development 119:199-206
(1993); Niswander, L. et al., "FGF-4 replaces the apical ectodermal
ridge and directs outgrowth and patterning of the limb," Cell
75:579-587 (1993)), anterior-posterior axis by Sonic hedgehog
(Riddle, R. D. et al, "Sonic hedgehog mediates the polarizing
activity of the ZPA," Cell 75:1401-1416 (1993)), and the dorsal
ventral axis by wingless (Parr, B. A. et al., "Mouse Wnt genes
exhibit discrete domains of expression in the early embryonic CNS
and limb buds," Development 119:247-261 (1993); Riddle, R. D. et
al., "Induction of the LIM homeobox gene Lmx1 by Wnt7a establishes
dorsoventral pattern in the vertebrate limb," Cell 83:631-640
(1995); Vogel, A. et al., "Dorsal cell fate specified by chick Lmx1
during vertebrate limb development," Nature 378:716-720 (1995)).
These factors are closely cross-regulated in development. The
secretion of Wnt (wingless) is stimulated by Sonic hedgehog (SHH)
signaling and conversely the expression of SHH is supported by the
continued presence of wingless. SHH in turn influences fibroblast
growth factor (FGF) expression (Niswander, L. et al., "A positive
feedback loop coordinates growth and patterning in the vertebrate
limb," Nature 371:609-612 (1994); Niswander, L., et al., "Function
of FGF-4 in limb development," Mol Reprod Dev 39:83-88; discussion
88-89 (1994); Laufer, E. et al., "Sonic hedgehog and Fgf-4 act
through a signaling cascade and feedback loop to integrate growth
and patterning of the developing limb bud," Cell 79:993-1003
(1994)). Wingless is a ligand for a G-coupled protein receptor
named frizzled, which mediates a complex signaling cascade (Vinson,
C. R. and Adler, P. N., "Directional non-cell autonomy and the
transmission of polarity information by the frizzled gene of
Drosophila," Nature 329:549-551 (1987)). Transcriptional regulation
is also mediated by SHH cell surface interaction with its ligand,
Patched. Patched tonically inhibits signaling through Smoothened
until it binds to SHH. These pathways are illustrated in FIG. 1,
which has been adapted from reviews by others (Hunter, T.,
"Oncoprotein networks," Cell 88:333-346 (1997); Ng, J. K. et al.,
"Molecular and cellular basis of pattern formation during
vertebrate limb development," Curr Top Dev Biol 41:37-66 (1999);
Ramsdell, A. F. and Yost, H. J., "Molecular mechanisms of
vertebrate left-right development," Trends Genet 14:459-465
(1998)).
[0006] Head and neck squamous cell carcinoma (HNSCC) is the sixth
most common cancer in developed countries, and of the 44,000 annual
cases reported in the United States approximately 11,000 will
result in an unfavorable outcome (Landis, S. H. et al., "Cancer
statistics," CA Cancer J Clin. 49, 8-31 (1999); Parkin, D. M. et
al., "Global cancer statistics," CA Cancer J Clin. 49, 33-64
(1999)). Although metastatic HNSCC can respond to chemotherapy and
radiotherapy, it is seldom adequately controlled. Therefore, it is
important to identify new molecular determinants on HNSCC that may
be potential targets for chemotherapy or immunotherapy.
[0007] In APC-deficient colon carcinoma, beta-catenin accumulates
and is constitutively complexed with nuclear Tcf-4 (Sparks, A. B.
et al., "Mutational analysis of the APC/beta-catenin/Tcf pathway in
colorectal cancer," Cancer Res 58:1130-1134 (1998)). Other colon
carcinomas and melanomas also contain constitutive nuclear
Tcf-4/beta-catenin complexes as a result of mutations in the N
terminus of beta-catenin that render it insensitive to
downregulation by APC, and GSK3 beta (Morin, P. J. et al.,
"Activation of beta-catenin-Tcf signaling in colon cancer by
mutations in beta-catenin or APC," Science 275:1787-1790 (1997);
Rubinfeld, B. et al. "Stabilization of beta-catenin by genetic
defects in melanoma cell lines," Science 275:1790-1792 (1997)).
This results in the unregulated expression of Tcf-4 oncogenic
target genes, such as c-myc, cyclin D1, and c-jun (He, T. C. et
al., "Identification of C-MYC as a target of the APC pathway,"
Science 281:1509-1512 (1998); Shtutman, M. et al., "The cyclin D1
gene is a target of the beta-catenin/LEF-1 pathway," Proc. Nat'l.
Acad Sci. USA 96:5522-5527 (1999); Li, L. et al., "Disheveled
proteins lead to two signaling pathways. Regulation of LEF-1 and
c-Jun N-terminal kinase in mammalian cells," J Biol Chem
274:129-134 (1999)). The expression of covalently linked
beta-catenin and LEF-1 has been directly demonstrated to result in
the oncogenic transformation of chicken fibroblasts (Aoki, M. et
al, "Nuclear endpoint of Wnt signaling: neoplastic transformation
induced by transactivating lymphoid-enhancing factor 1," Proc.
Nat'l. Acad. Sci. USA 96:139-144 (1999)). Similar mechanisms
leading to deregulation of Tcf target gene activity are likely to
be involved in melanoma (Rimm, D. L. et al., "Frequent
nuclear/cytoplasmic localization of beta-catenin without exon 3
mutations in malignant melanoma," Am J Pathol 154:325-329 (1999)),
breast cancer (Bui, T. D. et al., "A novel human Wnt gene, WNT10B,
maps to 12q13 and is expressed in human breast carcinomas,"
Oncogene 14:1249-1253 (1997)), heptocellular carcinoma (de La
Coste, A. et al., "Somatic mutations of the beta-catenin gene are
frequent in mouse and human heptocellular carcinomas," Proc Nat'l.
Acad. Sci. USA 95:8847-8851 (1998)), ovarian cancer (Palacios, J.,
and Gamallo, C., "Mutations in the beta-catenin gene (CTNNB1) in
endometrioid ovarian carcinomas," Cancer Res 58:1344-1347 (1998)),
endometrial cancer (Ikeda, T., "Mutational analysis of the CTNNB1
(beta-catenin) gene in human endometrial cancer: frequent mutations
at codon 34 that cause nuclear accumulation," Oncol Rep 7:323-326
(2000)), medulloblastoma (Hamilton, S. R. et al., "The molecular
basis of Turcot's syndrome," N. Engl J Med 332:839-847 (1995)),
pilomatricomas (Chan, E. F. et al. "A common human skin tumour is
caused by activating mutations in beta-catenin," Nat. Genet
21:410-413 (1999)), and prostate cancer (Iozzo, R. V. et al.,
"Aberrant expression of the growth factor Wnt-5A in human
malignancy," Cancer Res 55:3495-3499 (1995)).
[0008] Other growth regulation pathways in tumors have also
attracted recent interest. Many epithelial tumors express excess
amounts of epidermal growth factor-receptor tyrosine kinases,
particularly epidermal growth factor receptor (EGFR, or ErbB-1),
and HER2 (ErbB-2) (Coussens, L. et al, "Tyrosine kinase receptor
with extensive homology to EGF receptor shares chromosomal location
with neu oncogene," Science 230:1132-1139 (1985); King, C. R. et
al., "Amplification of a novel v-erbB-related gene in a human
mammary carcinoma," Science 229:974-976 (1985)). HER2 is
transmembrane tyrosine kinase receptor, which dimerizes with
another member of the EGFR family to form an active dimeric
receptor (Akiyama, T. et al., "The product of the human c-erbB-2
gene: a 185-kilodalton glycoprotein with tyrosine kinase activity,"
Science 232:1644-1646 (1986)). The resulting phosphorylation of
tyrosine residues initiates complex signaling pathways that
ultimately lead to cell division. HER2 is overexpressed in 25 to 30
percent of breast cancers, usually as a result of gene
amplification (Slamon, D. J. et al., "Studies of the HER-2/neu
proto-oncogene in human breast and ovarian cancer," Science
244:707-712 (1989)). A high level of this protein is associated
with an adverse prognosis (Slamon, D. J. et al., "Human breast
cancer: correlation of relapse and survival with amplification of
the HER-2/neu oncogene," Science 235:177-182 (1987); Ravdin, P. M.
and Chamness, G. C., "The c-erbB-2 proto-oncogene as a prognostic
and predictive marker in breast cancer: a paradigm for the
development of other macromolecular markers--a review," Gene
159:19-27 (1995)).
[0009] In the past decade there has been tremendous progress in
identifying genetic and molecular changes that occur during the
transformation of malignant cells. Many malignant cells have a less
differentiated phenotype, and a higher growth fraction than normal
in adult tissues. These basic characteristics are similar to
immature or embryonic cells. During the development of the embryo,
various cell surface receptors and ligands direct tissue pattern
formation, and cellular differentiation (Hunter, T., "Oncoprotein
networks," Cell 88, 333-346 (1997); Ng, J. K. et al., "Molecular
and cellular basis of pattern formation during vertebrate limb
development," Curr Top Dev Biol. 41, 37-66 (1999); Ramsdell, A. F.
and Yost, H. J., "Molecular mechanisms of vertebrate left-right
development," Trends Genet. 14, 459-465 (1998)). The expression of
these receptors and ligands is often no longer required in fully
matured adult tissues. Because they are expressed on the cell
surface, the receptors and ligands important for morphologic
patterning and tissue differentiation could be targets for the
immunotherapy of tumors that have arisen from residual immature
cells, or that have undergone de-differentiation.
[0010] Genes of the wingless (Wnt) and frizzled (Fzd) class have an
established role in cell morphogenesis and cellular differentiation
(Parr, B. A. et al., "Mouse Wnt genes exhibit discrete domains of
expression in the early embryonic CNS and limb buds," Development,
119, 247-261 (1993); Riddle, R. D. et al., "Induction of the LIM
homeobox gene Lmx1 by WNT7a establishes dorsoventral pattern in the
vertebrate limb," Cell 83, 631-640 (1995); Vogel, A. et al., (1995)
"Dorsal cell fate specified by chick Lmx1 during vertebrate limb
development," Nature 378, 716-720 (1995)). The Wnt proteins are
extracellular ligands for the Fzd receptors, which resemble typical
G protein coupled receptors (GPCRs). The first member of the 19
known human Wnt genes, Wnt-1, was initially discovered because of
its oncogenic properties (Nusse, R. and Varmus, H. E., "Many tumors
induced by the mouse mammary tumor virus contain a provirus
integrated in the same region of the host genome," Cell 31, 99-109
(1982)). The Wnt glycoproteins bind to one or more of the 10 known,
7 transmembrane domain G-protein coupled Fzd receptors, to initiate
a chain of signaling events that often culminates in the
stabilization and nuclear translocation of .beta.-catenin, with
resultant heterodimerization with one of the four members of the
LEF/TCF family of transcription factors (Cadigan, K. M. and Nusse,
R., "Wnt signaling: a common theme in animal development," Genes
Dev., 11, 3286-3305 (1997); Miller, J. R. et al., "Mechanism and
function of signal transduction by the Wnt/.beta.-catenin and
Wnt/Ca2+ pathways," Oncogene 18, 7860-7872 (1999)). These
transcription factor complexes control the activities of specific
Wnt target genes, including developmental regulators and other
genes involved in coordinating cell proliferation, cell-cell
interactions, and cell-matrix interactions (Vogel, A. and Tickle,
C., "FGF-4 maintains polarizing activity of posterior limb bud
cells in vivo and in vitro," Development 119:199-206 (1993)). The
overexpression of .beta.-catenin and LEF-1 has been demonstrated to
result in the oncogenic transformation of chicken fibroblasts
(Aoki, M. et al., "Nuclear endpoint of Wnt signaling: neoplastic
transformation induced by transactivating lymphoid-enhancing factor
1," Proc. Nat'l. Acad. Sci. USA 96, 139-144 (1999)).
[0011] A recent survey using microarray techniques showed that most
HNSCC overexpress mRNAs of the Wnt family (Leethanakul, C. et al.,
"Distinct pattern of expression of differentiation and
growth-related genes in squamous cell carcinomas of the head and
neck revealed by the use of laser capture microdissection and cDNA
arrays," Oncogene 19, 3220-3224 (2000)). However, the various Wnt
mRNAs are very homologous, and hybridization in microarrays often
cannot distinguish between closely related templates.
[0012] A murine monoclonal antibody 4DS binds with high affinity to
the extracellular domain of HER2, thereby blocking its function in
signal transduction (Hudziak, R. M. et al. "p185HER2 monoclonal
antibody has antiproliferative effects in vitro and sensitizes
human breast tumor cells to tumor necrosis factor," Mol Cell Biol
9:1165-1172 (1989); Fendly, B. M. et al. "Characterization of
murine monoclonal antibodies reactive to either the human epidermal
growth factor receptor or HER2/neu gene product," Cancer Res
50:1550-1558 (1990); Fendly, B. M. et al. "The extracellular domain
of HER2/neu is a potential immunogen for active specific
immunotherapy of breast cancer," J Biol Response Mod 9:449-455
(1990)). In experimental models of breast cancer, it was active in
vitro and in vivo, and had greater anti-tumor effects when combined
with chemotherapy Hudziak, R. M. et al. "p185HER2 monoclonal
antibody has antiproliferative effects in vitro and sensitizes
human breast tumor cells to tumor necrosis factor," Mol Cell Biol
9:1165-1172 (1989); Pietras, R. J. et al., "Antibody to HER-2/neu
receptor blocks DNA repair after cisplatin in human breast and
ovarian cancer cells," Oncogene 9:1829-1838 (1994). A recently
completed phase 3 randomized clinical trial of a humanized form of
4DS monoclonal antibody, trastuzumab (Herceptin; Genentech, Inc,
South San Francisco, Calif.), demonstrated efficacy against some
forms of breast tumors overexpressing HER2 (Slamon, D. J. et al.,
"Use of chemotherapy plus a monoclonal antibody against HER2 for
metastatic breast cancer that overexpresses HER2," N Engl J Med
344:783-792 (2001).
SUMMARY OF THE INVENTION
[0013] The present invention provides methods to identify specific
Wnt and/or Fzd proteins that are overexpressed in cancer cells.
Overexpression can refer to increased levels of particular Wnt
and/or Fzd protein levels in cancers cells releative to levels of
the same Wnt and/or Fzd protein in non-cancer cells of the same
tissue type. Alternatively, overexpression can refer to increased
levels of particular Wnt and/or Fzd levels in cancers cells
relative to levels of different Wnt and/or Fzd proteins in the same
cancer cells. Additionally in some cancers, the Wnt and/or Fzd
protein will be overexpressed when compared to both the same Wnt
and/or Fzd protein in a non-cancer cells of the same tissue type,
and different Wnt and/or Fzd proteins in the same cancer cells.
[0014] In one aspect, the present invention provides a method of
inhibiting the proliferation or survival of breast cancer cells, in
breast cancer cells that overexpress a Wnt protein in a Wnt/Fzd
signaling pathway when compared to non-cancer cells. The Wnt
protein can be Wnt7b, Wnt-10b, or Wnt-14. The breast cancer cells
are contacted with an agent that inhibits the Wnt/Fzd signaling
pathway in the cancer cells. In some embodiments, the agent is an
antagonist of the Wnt/Fzd signaling pathway. In preferred
embodiments, agent is an anti-Wnt antibody that specifically binds
Wnt7b, Wnt-10b, or Wnt-14. In a further embodiment, the anti-Wnt
antibody facilitates cellular toxicity or killing by complement. In
another aspect of the invention, the Wnt protein is overexpressed
when compared to another Wnt protein in the same cancer cells. In a
further aspect, the Wnt protein is required for proliferation or
survival of the cancer cell.
[0015] The invention also provides a method of treating a patient
with a breast cancer, where the cancer cells overexpress a Wnt
protein in a Wnt/Fzd signaling pathway when compared to non-cancer
cells. The Wnt protein can be Wnt7b, Wnt-10b, or Wnt-14. The breast
cancer cells are contacted with an agent that inhibits the Wnt/Fzd
signaling pathway in the cancer cells. In some embodiments, the
agent is an antagonist of the Wnt/Fzd signaling pathway. In
preferred embodiments, agent is an anti-Wnt antibody that
specifically binds Wnt7b, Wnt-10b, or Wnt-14. In a further
embodiment, the anti-Wnt antibody facilitates cellular toxicity or
killing by complement. In another aspect of the invention, the Wnt
protein is overexpressed when compared to another Wnt protein in
the same cancer cells. In a further aspect, the Wnt protein is
required for proliferation or survival of the cancer cell.
[0016] In one aspect, the invention provides a method of inhibiting
the proliferation or survival of chronic lymphocytic leukemia cells
that overexpress a Wnt protein in a Wnt/Fzd signaling pathway when
compared to non-cancer cells. The Wnt protein can be Wnt3 and
Wnt-16. The chronic lymphocytic leukemia cells are contacted with
an agent that inhibits the Wnt/Fzd signaling pathway in the cancer
cells. In some embodiments, the agent is an antagonist of the
Wnt/Fzd signaling pathway. In preferred embodiments, the agent is
an anti-Wnt antibody that specifically binds Wnt3, or Wnt-16. In a
further embodiment, the anti-Wnt antibody facilitates cellular
toxicity or killing by complement. In another aspect of the
invention, the Wnt protein is overexpressed when compared to
another Wnt protein in the same cancer cells. In a further aspect,
the Wnt protein is required for proliferation or survival of the
cancer cell.
[0017] The invention also provides, a method of treating a patient
with chronic lymphocytic leukemia, where the chronic lymphocytic
leukemia cells overexpress a Wnt protein in a Wnt/Fzd signaling
pathway when compared to non-cancer cells. The Wnt protein can be
Wnt3 and Wnt-16. The chronic lymphocytic leukemia cells are
contacted with an agent that inhibits the Wnt/Fzd signaling pathway
in the cancer cells. In some embodiments, the agent is an
antagonist of the Wnt/Fzd signaling pathway. In preferred
embodiments, the agent is an anti-Wnt antibody that specifically
binds Wnt3, or Wnt-16. In a further embodiment, the anti-Wnt
antibody facilitates cellular toxicity or killing by complement. In
another aspect of the invention, the Wnt protein is overexpressed
when compared to another Wnt protein in the same cancer cells. In a
further aspect, the Wnt protein is required for proliferation or
survival of the cancer cell.
[0018] In one aspect, the invention provides a method of inhibiting
the proliferation or survival of mantle zone lymphoma cells that
overexpress a Wnt protein in a Wnt/Fzd signaling pathway when
compared to non-cancer cells. The Wnt protein can be Wnt-16. The
mantle zone lymphoma cells are contacted with an agent that
inhibits the Wnt/Fzd signaling pathway in the cancer cells. In some
embodiments, the agent is an antagonist of the Wnt/Fzd signaling
pathway. In preferred embodiments, the agent is an anti-Wnt
antibody that specifically binds Wnt-16. In a further embodiment,
the anti-Wnt antibody facilitates cellular toxicity or killing by
complement. In another aspect of the invention, the Wnt protein is
overexpressed when compared to another Wnt protein in the same
cancer cells. In a further aspect, the Wnt protein is required for
proliferation or survival of the cancer cell.
[0019] The invention also provides, a method of treating a patient
with mantle zone lymphoma, when the mantle zone lymphoma cells
overexpress a Wnt protein in a Wnt/Fzd signaling pathway when
compared to non-cancer cells. The Wnt protein can be Wnt-16. The
mantle zone lymphoma cells are contacted with an agent that
inhibits the Wnt/Fzd signaling pathway in the cancer cells. In some
embodiments, the agent is an antagonist of the Wnt/Fzd signaling
pathway. In preferred embodiments, the agent is an anti-Wnt
antibody that specifically binds Wnt-16. In a further embodiment,
the anti-Wnt antibody facilitates cellular toxicity or killing by
complement. In another aspect of the invention, the Wnt protein is
overexpressed when compared to another Wnt protein in the same
cancer cells. In a further aspect, the Wnt protein is required for
proliferation or survival of the cancer cell.
[0020] In one aspect, the present invention provides a method of
inhibiting the proliferation or survival of breast cancer cells
that overexpress a Fzd protein in a Wnt/Fzd signaling pathway when
compared to non-cancer cells. The Fzd protein can be Fzd3, Fzd4,
Fzd6, Fzd7, or Fzd10. The breast cancer cells are contacted with an
agent that inhibits the Wnt/Fzd signaling pathway in the cancer
cells. In some embodiments, the agent is an antagonist of the
Wnt/Fzd signaling pathway. In preferred embodiments, the agent is
an anti-Fzd antibody that specifically binds Fzd3, Fzd4, Fzd6,
Fzd7, or Fzd10. In a further embodiment, the anti-Fzd antibody
facilitates cellular toxicity or killing by complement. In another
aspect, the Fzd protein is overexpressed when compared to another
Fzd protein in the same cancer cells. In a further aspect, wherein
the Fzd protein is required for proliferation or survival of the
cancer cell.
[0021] The invention also provides a method of treating a patient
with a breast cancer, where the breast cancer cells overexpress a
Wnt protein when compared to non-cancer cells. The Fzd protein can
be Fzd3, Fzd4, Fzd6, Fzd7, or Fzd10. The breast cancer cells are
contacted with an agent that inhibits the Wnt/Fzd signaling pathway
in the cancer cells. In some embodiments, the agent is an
antagonist of the Wnt/Fzd signaling pathway. In preferred
embodiments, the agent is an anti-Fzd antibody that specifically
binds Fzd3, Fzd4, Fzd6, Fzd7, or Fzd10. In a further embodiment,
the anti-Fzd antibody facilitates cellular toxicity or killing by
complement. In another aspect, the Fzd protein is overexpressed
when compared to another Fzd protein in the same cancer cells. In a
further aspect, wherein the Fzd protein is required for
proliferation or survival of the cancer cell.
[0022] In one aspect, the invention provides a method of inhibiting
the proliferation or survival of chronic lymphocytic leukemia cells
that overexpress a Fzd protein in a Wnt/Fzd signaling pathway when
compared to non-cancer cells. The Fzd protein can be Fzd3. The
chronic lymphocytic leukemia cells are contacted with an agent that
inhibits the Wnt/Fzd signaling pathway in the chronic lymphocytic
leukemia cells. In some embodiments, the agent is an antagonist of
the Wnt/Fzd signaling pathway. In a preferred embodiment, the agent
is an anti-Fzd antibody that specifically binds Fzd3. In a further
embodiment, the anti-Fzd antibody facilitates cellular toxicity or
killing by complement. In another aspect, the Fzd protein is
overexpressed when compared to another Fzd protein in the same
cancer cells. In a further aspect, wherein the Fzd protein is
required for proliferation or survival of the cancer cell.
[0023] The invention also provides a method of treating a patient
with chronic lymphocytic leukemia, wherein the chronic lymphocytic
leukemia cells overexpress a Fzd protein in a Wnt/Fzd signaling
pathway when compared to non-cancer cells. The Fzd protein can be
Fzd3. The chronic lymphocytic leukemia cells are contacted with an
agent that inhibits the Wnt/Fzd signaling pathway in the chronic
lymphocytic leukemia cells. In some embodiments, the agent is an
antagonist of the Wnt/Fzd signaling pathway. In a preferred
embodiment, the agent is an anti-Fzd antibody that specifically
binds Fzd3. In a further embodiment, the anti-Fzd antibody
facilitates cellular toxicity or killing by complement. In another
aspect, the Fzd protein is overexpressed when compared to another
Fzd protein in the same cancer cells. In a further aspect, wherein
the Fzd protein is required for proliferation or survival of the
cancer cell.
[0024] In one aspect the present invention provides a method of
inhibiting the proliferation or survival of cancer cells that
overexpress a Wnt protein when compared to non-cancer cells, and
that also overexpress a downstream wnt/fzd regulated gene product
compared to non-cancer cells. The cancer cells are contacted with
an agent that inhibits the Wnt/Fzd signaling pathway in the cancer
cells. In some embodiments, the agent is an antibody directed
against the overexpressed Wnt protein. In a further embodiment, the
Wnt protein is also overexpressed when compared to another Wnt
protein in the same cancer cells. In another embodiment, Wnt
protein is required for proliferation or survival of the cancer
cell. As an example, the proliferation of breast cancer cells that
overexpresses Wnt7b, Wnt-10b, or Wnt-14 and also overexpresses
cyclin D1, c-myc, and WISP family member can be inhibited by an
antibody that binds specifically to Wnt7b, Wnt-10b, or Wnt-14.
[0025] The invention also provides a method of treating a patient
with a cancer containing cells that that overexpress a Wnt protein
when compared to non-cancer cells, and that also overexpress a
downstream wnt/fzd regulated gene product compared to non-cancer
cells. The cancer cells are contacted with an agent that inhibits
the Wnt/Fzd signaling pathway in the cancer cells. In some
embodiments, the agent is an antibody directed against the
overexpressed Wnt protein. In a further embodiment, the Wnt protein
is also overexpressed when compared to another Wnt protein in the
same cancer cells. In another embodiment, Wnt protein is required
for proliferation or survival of the cancer cell. As an example, a
patient with breast cancer containing cells that overexpresses
Wnt7b, Wnt-10b, or Wnt-14 and also overexpresses cyclin D1, c-myc,
and WISP family member can be inhibited by an antibody that binds
specifically to Wnt7b, Wnt-10b, or Wnt-14.
[0026] In one aspect the present invention provides a method of
inhibiting the proliferation or survival of cancer cells that
overexpress a Fzd protein when compared to non-cancer cells, and
that also overexpress a downstream wnt/fzd regulated gene product
compared to non-cancer cells. The cancer cells are contacted with
an agent that inhibits the Wnt/Fzd signaling pathway in the cancer
cells. In some embodiments, the agent is an antibody directed
against the overexpressed Fzd protein. In a further embodiment, the
Fzd protein is also overexpressed when compared to another Fzd
protein in the same cancer cells. In another embodiment, Fzd
protein is required for proliferation or survival of the cancer
cell. As an example, the proliferation of breast cancer cells that
overexpresses Fzd3, Fzd4, Fzd6, Fzd7, or Fzd10 and that also
overexpresses cyclin D1, c-myc, and WISP family member can be
inhibited by an antibody that binds specifically to Fzd3, Fzd4,
Fzd6, Fzd7, or Fzd10.
[0027] The invention also provides a method of treating a patient
with a cancer containing cells that that overexpress a Fzd protein
when compared to non-cancer cells, and that also overexpress a
downstream wnt/fzd regulated gene product compared to non-cancer
cells. The cancer cells are contacted with an agent that inhibits
the Wnt/Fzd signaling pathway in the cancer cells. In some
embodiments, the agent is an antibody directed against the
overexpressed Fzd protein. In a further embodiment, the Fzd protein
is also overexpressed when compared to another Fzd protein in the
same cancer cells. In another embodiment, Fzd protein is required
for proliferation or survival of the cancer cell. As an example, a
patient with breast cancer containing cells that overexpresses
Fzd3, Fzd4, Fzd6, Fzd7, or Fzd10 and also overexpresses cyclin D1,
c-myc, and WISP family member can be inhibited by an antibody that
binds specifically to Fzd3, Fzd4, Fzd6, Fzd7, and Fzd10.
[0028] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Wnt-1 encoding nucleic acid, and specifically
hybridizes to the same sequence the polynucleotides:
5'-CGAACCTGCTTACAGACTCCAA-3' and 5'-CAGACGCCGCTGTTTGC-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Wnt-1 in a
biological sample, by contacting the sample with the isolated
polynucleotide so that hybridization with the Wnt-1 nucleic acid
can be detected.
[0029] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Wnt-2 encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-GGATGACCAAGTGTGGGTGTAAG-3- ' and 5'-GTGCACATCCAGAGCTTCCA-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Wnt-2 in a
biological sample, by contacting the sample with the isolated
polynucleotide so that hybridization with the Wnt-2 encoding
nucleic acid present can be detected.
[0030] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Wnt-2b encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-GGCACGAGTGATCTGTGACAATA-3- ' and 5'-CGCATGATGTCTGGGTAACG-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Wnt-2b in a
biological sample, by contacting the sample with the isolated
polynucleotide so that specific hybridization with the Wnt-2b
encoding nucleic acid present can be detected.
[0031] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Wnt-3 encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-CTGGGCCAGCAGTACACATCT-3' and 5'-GGCATGATCTCGATGTAATTGC-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Wnt-3 in a
biological sample by contacting the sample with the isolated
polynucleotide ao that specific hybridization with the Wnt-3
encoding nucleic acid present can be detected.
[0032] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Wnt-3a encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-CCCGTGCTGGACAAAGCT-3' and 5'-TCTGCACATGAGCGTGTCACT-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Wnt-3a in a
biological sample, by contacting the sample with the isolated
polynucleotide so that specific hybridization with the Wnt-3a
encoding nucleic acid present can be detected.
[0033] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Wnt-4 encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-GGAGGAGACGTGCGAGAAAC-3' and 5'-CAGGTTCCGCTTGCACATCT-3' The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Wnt-4 in a
biological sample, by contacting the sample with the isolated
polynucleotide so that specific hybridization with the Wnt-4
encoding nucleic acid present can be detected.
[0034] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Wnt-5a encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-TCTCCTTCGCCCAGGTTGTA-3' and 5'-CTTCTGACATCTGAACAGGGTTATTC-3'.
The invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Wnt-5a in a
biological sample, by contacting the sample with the isolated
polynucleotide so that specific hybridization with the Wnt-5a
encoding nucleic acid present can be detected.
[0035] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Wnt-5b encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-CCAACTCCTGGTGGTCATTAGC-3' and 5'-TGGGCACCGATGATAAACATC-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Wnt-5b in a
biological sample, by contacting the sample with the isolated
polynucleotide of claim 99, under conditions to permit so that
specific hybridization with the Wnt-5b encoding nucleic acid
present can be detected.
[0036] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Wnt-6 encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-TCCGCCGCTGGAATTG-3' and 5'-AGGCCGTCTCCCGAATGT-3'. The invention
also provides a method of specifically detecting the presence or
absence of a nucleic acid encoding Wnt-6 in a biological sample, by
contacting the sample with the isolated polynucleotide so that
specific hybridization with the Wnt-6 encoding nucleic acid present
can be detected.
[0037] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Wnt-7a encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-GACGCCATCATCGTCATAGGA-3' and 5'-GGCCATTGCGGAACTGAA-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Wnt-7a in a
biological sample, by comprising contacting the sample with the
isolated polynucleotide so that specific hybridization with the
Wnt-7a encoding nucleic acid present can be detected.
[0038] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Wnt-7b encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-TGAAGCTCGGAGCACTGTCA-3' and 5'-GGCCAGGAATCTTGTTGCA-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Wnt-7b in a
biological sample, by contacting the sample with the isolated
polynucleotide so that specific hybridization with the Wnt-7b
encoding nucleic acid present can be detected.
[0039] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Wnt-8a encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-GCAGAGGCGGAACTGATCTT-3' and 5'-CGACCCTCTGTGCCATAGATG-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Wnt-8a in a
biological sample, by contacting the sample with the isolated
polynucleotide so that specific hybridization with the Wnt-8a
encoding nucleic acid present can be detected.
[0040] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Wnt-8b encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-AATCGGGAGACAGCATTTGTG-3' and 5'-ATCTCCAAGGCTGCAGTTTCTAGT-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Wnt-8b in a
biological sample, by contacting the sample with the isolated
polynucleotide so that specific hybridization with the Wnt-8b
encoding nucleic acid present can be detected.
[0041] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Wnt-10a encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-CTGGGTGCTCCTGTTCTTCCTA-3' and 5'-GAGGCGGAGGTCCAGAATG-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Wnt-10a in a
biological sample, by contacting the sample with the isolated
polynucleotide so that specific hybridization with the Wnt-10a
encoding nucleic acid present can be detected.
[0042] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Wnt-10b encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-CCTCGCGGGTCTCCTGTT-3' and 5'-AGGCCCAGAATCTCATTGCTTA-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Wnt-10b in a
biological sample, by contacting the sample with the isolated
polynucleotide so that specific hybridization with the Wnt-10b
encoding nucleic acid present can be detected.
[0043] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Wnt-11 encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-CGTGTGCTATGGCATCAAGTG-3' and 5'-GCAGTGTTGCGTCTGGTTCA-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Wnt-11 in a
biological sample, by contacting the sample with the isolated
polynucleotide so that specific hybridization with the Wnt-11
encoding nucleic acid present can be detected.
[0044] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Wnt-14 encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides
5'-GGGCAGACGGTCAAGCAA-3' and 5'-CCAGCCTTGATCACCTTCACA-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Wnt-14 in a
biological sample, by contacting the sample with the isolated
polynucleotides so that specific hybridization with the Wnt-14
encoding nucleic acid present can be detected.
[0045] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Wnt-16 encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-GCCAATTTGCCGCTGAAC-3' and 5'-CGGCAGCAGGTACGGTTT-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Wnt-16 in a
biological sample, by contacting the sample with the isolated so
that specific hybridization with the Wnt-16 encoding nucleic acid
present can be detected.
[0046] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Fzd1 encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-CACCTTGTGAGCCGACCAA-3' and 5'-CAGCACTGACCAAATGCCAAT-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Fzd1 in a biological
sample, by contacting the sample with the isolated polynucleotide
so that specific hybridization with the Fzd1 encoding nucleic acid
present can be detected.
[0047] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Fzd2 encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-TTTCTGGGCGAGCGTGAT-3' and 5'-AAACGCGTCTCCTCCTGTGA-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Fzd2 in a biological
sample, by contacting the sample with with the polynucleotide so
that specific hybridization with the Fzd2 encoding nucleic acid
present can be detected.
[0048] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Fzd3 encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-TGGCTATGGTGGATGATCAAAG-3' and 5'-TGGAGGCTGCCGTGGTA-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Fzd3 in a biological
sample, by contacting the sample with the isolated polynucleotide
so that specific hybridization with the Fzd3 encoding nucleic acid
present can be detected.
[0049] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Fzd4 encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-GGCGGCATGTGTCTTTCAGT-3' and 5'-GAATTTGCTGCAGTTCAGACTCTCT-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Fzd4 in a biological
sample, by contacting the sample with the isolated polynucleotide
so that specific hybridization with the Fzd4 encoding nucleic acid
present can be detected.
[0050] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Fzd5 encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-CGCGAGCACAACCACATC-3' and 5'-AGAAGTAGACCAGGAGGAAGACGAT-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Fzd5 in a biological
sample, by contacting the sample with the isolated polynucleotide
so that specific hybridization with the Fzd5 encoding nucleic acid
present can be detected.
[0051] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Fzd6 encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-ACAAGCTGAAGGTCATTTCCAAA-3- ' and 5'-GCTACTGCAGAAGTGCCATGAT-3'.
The invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Fzd6 in a biological
sample, by contacting the sample with the isolated polynucleotide
so that specific hybridization between Fzd6 encoding nucleic acid
present can be detected.
[0052] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Fzd7 encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-CAACGGCCTGATGTACTTTAAGG-3- ' and 5'-CATGTCCACCAGGTAGGTGAGA-3'.
The invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Fzd7 in a biological
sample, by contacting the sample with the isolated polynucleotide
so that specific hybridization with the Fzd7 encoding nucleic acid
present can be detected.
[0053] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Fzd8 encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-GCTCGGTCATCAAGCAACAG-3' and 5'-ACGGTGTAGAGCACGGTGAAC-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Fzd8 in a biological
sample, by contacting the sample with the isolated polynucleotide
so that specific hybridization with the Fzd8 encoding nucleic acid
present can be detected.
[0054] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Fzd9 encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-GCGCTCAAGACCATCGTCAT-3' and 5'-ATCCGTGCTGGCCACGTA-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Fzd9 in a biological
sample, by contacting the sample with the isolated polynucleotide
so that specific hybridization with the Fzd9 encoding nucleic acid
present can be detected.
[0055] In another aspect, the invention provides an isolated
polynucleotide of less than about 100 nucleotides that specifically
hybridizes to a Fzd10 encoding nucleic acid, and specifically
hybridizes to the same sequence as the polynucleotides:
5'-GCCGCCATCAGCTCCAT-3' and 5'-TCATGTTGTAGCCGATGTCCTT-3'. The
invention also provides a method of specifically detecting the
presence or absence of a nucleic acid encoding Fzd10 in a
biological sample, by contacting the sample with the isolated
polynucleotide so that specific hybridization between Fzd10
encoding nucleic acid present can be detected.
[0056] Definitions
[0057] The terms "Wnt protein" or "Wnt ligand" refer to a family of
mammalian proteins related to the Drosophila segment polarity gene,
wingless. In humans, the Wnt family of genes typically encode 38 to
43 kDa cysteine rich glycoproteins having hydrophobic signal
sequence, and a conserved asparagine-linked oligosaccharide
consensus sequence (see e.g., Shimizu et al Cell Growth Differ
8:1349-1358 (1997)). The Wnt family contains at least 19 mammalian
members. Exemplary Wnt proteins include Wnt-1, Wnt-2, Wnt-2b (also
known as Wnt-13) Wnt-3, Wnt-3A, Wnt-4, Wnt-5A, Wnt-5B, Wnt-6,
Wnt-7A, Wnt-7B, Wnt-8A, Wnt-8B, Wnt-10A, Wnt-10B, Wnt-11, Wnt 14,
Wnt 15, and Wnt 16. The sequence of exemplary wnt proteins are set
forth in the informal sequence listing. As explained below, certain
cancers are associated with particular Wnt proteins. For example,
head and neck squamous cell carcinoma cells are associated with
Wnt-5a, Wnt-7a, Wnt-10b, or Wnt-13. Glioblastoma is associated with
Wnt-1 or Wnt-10b. Burkitt lymphoma and chronic lymphocytic leukemia
are associated with Wnt-1 or Wnt-10b. Malignant lymphocytes
overexpress Wnt-6, Wnt-14, or Wnt-16. Breast cancer is associated
with overexpression of wnt5a, wnt7b, wnt10b, and wnt14.
[0058] The terms "frizzled protein" or "frizzled receptor" refer to
a family of mammalian proteins related to the Drosophila frizzled
genes, which play a role in the development of tissue polarity. The
Frizzled family comprises at least 10 mammalian genes. Exemplary
human Frizzled receptors include Frizzled1, Frizzled2, Frizzled3,
Frizzled4, Frizzled5, Frizzled6, Frizzled7, Frizzled8, Frizzled9
and Frizzled10. The sequence of exemplary Frizzled receptors are
set forth in the informal sequence listing. The mammalian
homologues of the Drosophila frizzled protein share a number of
common structural motifs. The N terminus located at the
extracellular membrane surface is followed by a signal sequence, a
domain of 120 amino acids with an invariant pattern of 10 cysteine
residues, and a highly divergent region of 40-100 largely variable
hydrophilic amino acids. Putative hydrophobic segments form seven
membrane-spanning helices linked by hydrophilic loops, ending with
the C terminus located at the intracellular face of the membrane.
The cysteine-rich domains (CRDs) and the transmembrane segments are
strongly conserved, suggesting a working model in which an
extracellular CRD is tethered by a variable linker region to a
bundle of seven membrane-spanning-helices. Frizzled protein
receptors are, therefore, involved in a dynamic model of
transmembrane signal transduction analogous to G-protein-coupled
receptors with amino-terminal ligand binding domains.
[0059] In addition to the Wnt ligands, a family of secreted
frizzled-related proteins (sFRPs) has been isolated. sFRPs appear
to function as soluble endogenous modulators of Wnt signaling by
competing with the membrane-spanning frizzled receptors for the
binding of secreted Wnt ligands. sFRPs, therefore, modulate
apoptosis susceptibility, exerting an antagonistic effect on
programmed cell death. sFRPs can either antagonize Wnt function by
binding the protein and blocking access to its cell surface
signaling receptor, or they can enhance Wnt activity by
facilitating the presentation of ligand to the frizzled receptors.
To date, sFRPs have not yet been linked causatively to cancer.
[0060] The term "agent" or grammatical equivalents as used herein
describes any molecule, either naturally occurring or synthetic,
e.g., protein (for example an antibody or sFRP), oligopeptide
(e.g., from about 5 to about 25 amino acids in length, preferably
from about 10 to 20 or 12 to 18 amino acids in length, preferably
12, 15, or 18 amino acids in length), small chemical molecule,
polysaccharide, lipid (e.g., a sphingolipid), fatty acid,
polynucleotide, oligonucleotide, etc., that directly or indirectly
inhibits a Wnt/Fzd signaling pathway.
[0061] The terms "antagonists" or "inhibitors" of Wnt signaling or
of the wnt/Fzd signaling pathway refer to compounds that, e.g.,
bind to Wnt or Frizzled proteins, or partially or totally block or
inhibit Wnt/Fzd signaling as measured in known assays for Wnt/Fzd
signaling (e.g., measurement of .beta. catenin levels, or oncogene
expression controlled by Tcf and Lef transcription factors or other
downstream wnt/fzd regulated gene products). Inhibitors, include
antibodies directed against Wnt or Fzd proteins, and modified
versions of Wnt or Frizzled proteins, as well as naturally
occurring and synthetic ligands, antagonists, agonists, antibodies,
small chemical molecules, and the like. Assays for detecting
inhibitors or agaonists of the invention are described in more
detail below.
[0062] A "cancer cell that overexpresses a Wnt or Frizzled protein"
is a cancer cell in which expression of a particular Wnt protein is
at least about 2 times, usually at least about 5 times the level of
expression in a non-cancer cell from the same tissue type. In some
embodiments, wnt and/or fzd expression in a cancer cell is compared
to wnt and/or fzd expression in a non-cancer cell of a different
tissue-type or a panel of non-cancer cells of a different tissue
type. In addition, expression of particular Wnt and/or Frizzled
proteins can be compared to other Wnt and/or Frizzled proteins in
the same cell. Those proteins that are overexpressed in cancer
cells compared to non-cancer cells and that are overexpressed
compared to other Wnt and/or Frizzled proteins in the same cancer
cell are generally preferred. Methods for determining the level of
expression of a particular gene are well known in the art. Such
methods include RT-PCR, real time PCR, use of antibodies against
the gene products, and the like.
[0063] The terms "wnt signaling", "wnt/fzd" signaling and "fzd
signaling" are used interchangeably.
[0064] A "Wnt/Fzd signaling pathway" refers to activation of an
intracellular signal transduction pathway that is initiated by an
interaction between a specific Wnt protein and a specific Fzd
protein. Generally, the Wnt/Fzd interaction will be binding of a
Wnt protein to a Fzd receptor, leading to activation of a signal
transduction pathway. In some instances activation of the Wnt/Fzd
signaling pathway will lead to induction of downstream wnt and/or
fzd inducible genes. A "downstream wnt/fzd regulated gene product"
is a protein or RNA that is upregulated, or otherwise regulated, as
a result of signaling by a wnt/fzd transduction pathway.
[0065] "Proliferation of a cancer cell" refers to cell division and
increase in the number of cancer cells. "Inhibition of
proliferation" refers to a decrease in the rate of proliferation
(e.g., cellular division), cessation of proliferation (e.g., entry
into G0 phase or senescense), or death of a cell, including
necrotic cell death.
[0066] "Inhibition of survival of a cancer cell" refers to
induction or relief of inhibition of a programmed cell death
process, e.g., apoptosis.
[0067] The term "contact" or "contacting" is used herein
interchangeably with the following: combined with, added to, mixed
with, passed over, incubated with, flowed over, etc.
[0068] "Nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form. Exemplary wnt and fzd nucleic acids are found
in the informal sequence listing. The term encompasses nucleic
acids containing known nucleotide analogs or modified backbone
residues or linkages, which are synthetic, naturally occurring, and
non-naturally occurring, which have similar binding properties as
the reference nucleic acid, and which are metabolized in a manner
similar to the reference nucleotides. Examples of such analogs
include, without limitation, phosphorothioates, phosphoramidates,
methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl
ribonucleotides, peptide-nucleic acids (PNAs).
[0069] Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions) and
complementary sequences, as well as the sequence explicitly
indicated. Thus, the terms "a Wnt encoding nuleic acid" and "a Fzd
encoding nucleic acid" include both coding and complementary
noncoding sequences. Specifically, degenerate codon substitutions
may be achieved by generating sequences in which the third position
of one or more selected (or all) codons is substituted with
mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic
Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem.
260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98
(1994)). The term nucleic acid is used interchangeably with gene,
cDNA, mRNA, oligonucleotide, and polynucleotide.
[0070] As used herein, "antibody" includes reference to an
immunoglobulin molecule immunologically reactive with a particular
antigen, and includes both polyclonal and monoclonal antibodies.
The term also includes genetically engineered forms such as
chimeric antibodies (e.g., humanized murine antibodies) and
heteroconjugate antibodies (e.g., bispecific antibodies). The term
"antibody" also includes antigen binding forms of antibodies,
including fragments with antigen-binding capability (e.g., Fab',
F(ab').sub.2, Fab, Fv and rIgG. See also, Pierce Catalog and
Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.). See
also, e.g., Kuby, J., Immunology, 3.sup.rd Ed., W. H. Freeman &
Co., New York (1998). The term also refers to recombinant single
chain Fv fragments (scFv). The term antibody also includes bivalent
or bispecific molecules, diabodies, triabodies, and tetrabodies.
Bivalent and bispecific molecules are described in, e.g., Kostelny
et al. (1992) J Immunol 148:1547, Pack and Pluckthun (1992)
Biochemistry 31:1579, Hollinger et al., 1993, supra, Gruber et al.
(1994) J Immunol:5368, Zhu et al. (1997) Protein Sci 6:781, Hu et
al. (1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res.
53:4026, and McCartney, et al. (1995) Protein Eng. 8:301.
[0071] An antibody immunologically reactive with a particular
antigen can be generated by recombinant methods such as selection
of libraries of recombinant antibodies in phage or similar vectors,
see, e.g., Huse et al., Science 246:1275-1281 (1989); Ward et al.,
Nature 341:544-546 (1989); and Vaughan et al., Nature Biotech.
14:309-314 (1996), or by immunizing an animal with the antigen or
with DNA encoding the antigen.
[0072] Typically, an immunoglobulin has a heavy and light chain.
Each heavy and light chain contains a constant region and a
variable region, (the regions are also known as "domains"). Light
and heavy chain variable regions contain four "framework" regions
interrupted by three hypervariable regions, also called
"complementarity-determining regions" or "CDRs". The extent of the
framework regions and CDRs have been defined. The sequences of the
framework regions of different light or heavy chains are relatively
conserved within a species. The framework region of an antibody,
that is the combined framework regions of the constituent light and
heavy chains, serves to position and align the CDRs in three
dimensional space.
[0073] The CDRs are primarily responsible for binding to an epitope
of an antigen. The CDRs of each chain are typically referred to as
CDR1, CDR2, and CDR3, numbered sequentially starting from the
N-terminus, and are also typically identified by the chain in which
the particular CDR is located. Thus, a V.sub.H CDR3 is located in
the variable domain of the heavy chain of the antibody in which it
is found, whereas a V.sub.L CDR1 is the CDR1 from the variable
domain of the light chain of the antibody in which it is found.
[0074] References to "V.sub.H" or a "VH" refer to the variable
region of an immunoglobulin heavy chain of an antibody, including
the heavy chain of an Fv, scFv, or Fab. References to "V.sub.L" or
a "VL" refer to the variable region of an immunoglobulin light
chain, including the light chain of an Fv, scFv, dsFv or Fab.
[0075] The phrase "single chain Fv" or "scFv" refers to an antibody
in which the variable domains of the heavy chain and of the light
chain of a traditional two chain antibody have been joined to form
one chain. Typically, a linker peptide is inserted between the two
chains to allow for proper folding and creation of an active
binding site.
[0076] A "chimeric antibody" is an immunoglobulin molecule in which
(a) the constant region, or a portion thereof, is altered, replaced
or exchanged so that the antigen binding site (variable region) is
linked to a constant region of a different or altered class,
effector function and/or species, or an entirely different molecule
which confers new properties to the chimeric antibody, e.g., an
enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the
variable region, or a portion thereof, is altered, replaced or
exchanged with a variable region having a different or altered
antigen specificity.
[0077] A "humanized antibody" is an immunoglobulin molecule which
contains minimal sequence derived from non-human immunoglobulin.
Humanized antibodies include human immunoglobulins (recipient
antibody) in which residues from a complementary determining region
(CDR) of the recipient are replaced by residues from a CDR of a
non-human species (donor antibody) such as mouse, rat or rabbit
having the desired specificity, affinity and capacity. In some
instances, Fv framework residues of the human immunoglobulin are
replaced by corresponding non-human residues. Humanized antibodies
may also comprise residues which are found neither in the recipient
antibody nor in the imported CDR or framework sequences. In
general, a humanized antibody will comprise substantially all of at
least one, and typically two, variable domains, in which all or
substantially all of the CDR regions correspond to those of a
non-human immunoglobulin and all or substantially all of the
framework (FR) regions are those of a human immunoglobulin
consensus sequence. The humanized antibody optimally also will
comprise at least a portion of an immunoglobulin constant region
(Fc), typically that of a human immunoglobulin (Jones et al.,
Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329
(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)).
Humanization can be essentially performed following the method of
Winter and co-workers (Jones et al., Nature 321:522-525 (1986);
Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,
Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody.
Accordingly, such humanized antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species.
[0078] "Epitope" or "antigenic determinant" refers to a site on an
antigen to which an antibody binds. Epitopes can be formed both
from contiguous amino acids or noncontiguous amino acids juxtaposed
by tertiary folding of a protein. Epitopes formed from contiguous
amino acids are typically retained on exposure to denaturing
solvents whereas epitopes formed by tertiary folding are typically
lost on treatment with denaturing solvents. An epitope typically
includes at least 3, and more usually, at least 5 or 8-10 amino
acids in a unique spatial conformation. Methods of determining
spatial conformation of epitopes include, for example, x-ray
crystallography and 2-dimensional nuclear magnetic resonance. See,
e.g., Epitope Mapping Protocols in Methods in Molecular Biology,
Vol. 66, Glenn E. Morris, Ed (1996).
[0079] "Biological sample" as used herein is a sample of biological
tissue or fluid that contains nucleic acids or polypeptides, e.g.,
of a Wnt protein, polynucleotide or transcript. Such samples
include, but are not limited to, tissue isolated from primates,
e.g., humans, or rodents, e.g., mice, and rats. Biological samples
may also include sections of tissues such as biopsy and autopsy
samples, frozen sections taken for histologic purposes, blood,
plasma, serum, sputum, stool, tears, mucus, hair, skin, etc.
Biological samples also include explants and primary and/or
transformed cell cultures derived from patient tissues. A
biological sample is typically obtained from a eukaryotic organism,
most preferably a mammal such as a primate e.g., chimpanzee or
human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse;
rabbit; or a bird; reptile; or fish.
[0080] "Providing a biological sample" means to obtain a biological
sample for use in methods described in this invention. Most often,
this will be done by removing a sample of cells from an animal, but
can also be accomplished by using previously isolated cells (e.g.,
isolated by another person, at another time, and/or for another
purpose), or by performing the methods of the invention in vivo.
Archival tissues, having treatment or outcome history, will be
particularly useful.
[0081] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., about 60% identity, preferably 70%, 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher
identity over a specified region, when compared and aligned for
maximum correspondence over a comparison window or designated
region) as measured using a BLAST or BLAST 2.0 sequence comparison
algorithms with default parameters described below, or by manual
alignment and visual inspection (see, e.g., NCBI web site
http://www.ncbi.nlm.nih.gov/BLAST/or the like). Such sequences are
then said to be "substantially identical." This definition also
refers to, or may be applied to, the complement of a test sequence.
The definition also includes sequences that have deletions and/or
additions, as well as those that have substitutions, as well as
naturally occurring, e.g., polymorphic or allelic variants, and
man-made variants. As described below, the preferred algorithms can
account for gaps and the like. Preferably, identity exists over a
region that is at least about 25 amino acids or nucleotides in
length, or more preferably over a region that is 50-100 amino acids
or nucleotides in length.
[0082] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Preferably, default program parameters can be used,
or alternative parameters can be designated. The sequence
comparison algorithm then calculates the percent sequence
identities for the test sequences relative to the reference
sequence, based on the program parameters.
[0083] A "comparison window", as used herein, includes reference to
a segment of one of the number of contiguous positions selected
from the group consisting typically of from 20 to 600, usually
about 50 to about 200, more usually about 100 to about 150 in which
a sequence may be compared to a reference sequence of the same
number of contiguous positions after the two sequences are
optimally aligned. Methods of alignment of sequences for comparison
are well-known in the art. Optimal alignment of sequences for
comparison can be conducted, e.g., by the local homology algorithm
of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the
homology alignment algorithm of Needleman & Wunsch, J. Mol.
Biol. 48:443 (1970), by the search for similarity method of Pearson
& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
manual alignment and visual inspection (see, e.g., Current
Protocols in Molecular Biology (Ausubel et al., eds. 1995
supplement)).
[0084] Preferred examples of algorithms that are suitable for
determining percent sequence identity and sequence similarity
include the BLAST and BLAST 2.0 algorithms, which are described in
Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul
et al., J. Mol. Biol. 215:403-410 (1990). BLAST and BLAST 2.0 are
used, with the parameters described herein, to determine percent
sequence identity for the nucleic acids and proteins of the
invention. Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, e.g., for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) of 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0085] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin &
Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001. Log
values may be large negative numbers, e.g., 5, 10, 20, 30, 40, 40,
70, 90, 110, 150, 170, etc.
[0086] An indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the antibodies raised against the polypeptide encoded by the
second nucleic acid, wherein the antibodies are specific for the
polypeptide encoded by the second nucleic acid, as described below.
Thus, a polypeptide is typically substantially identical to a
second polypeptide, e.g., where the two peptides differ only by
conservative substitutions. Another indication that two nucleic
acid sequences are substantially identical is that the two
molecules or their complements hybridize to each other under
stringent conditions, as described below. Yet another indication
that two nucleic acid sequences are substantially identical is that
the same primers can be used to amplify the sequences.
[0087] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acids, but
to no other sequences. Stringent conditions are sequence-dependent
and will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide. For selective or specific
hybridization, a positive signal is at least two times background,
preferably 10 times background hybridization. Exemplary stringent
hybridization conditions can be as following: 50% formamide,
5.times.SSC, and 1% SDS, incubating at 42.degree. C., or,
5.times.SSC, 1% SDS, incubating at 65.degree. C., with wash in
0.2.times.SSC, and 0.1% SDS at 65.degree. C.
[0088] The terms "isolated," "purified," or "biologically pure"
refer to material that is substantially or essentially free from
components that normally accompany it as found in its native state.
Purity and homogeneity are typically determined using analytical
chemistry techniques such as polyacrylamide gel electrophoresis or
high performance liquid chromatography. A protein or nucleic acid
that is the predominant species present in a preparation is
substantially purified. In particular, an isolated nucleic acid is
separated from some open reading frames that naturally flank the
gene and encode proteins other than protein encoded by the gene.
The term "purified" in some embodiments denotes that a nucleic acid
or protein gives rise to essentially one band in an electrophoretic
gel. Preferably, it means that the nucleic acid or protein is at
least 85% pure, more preferably at least 95% pure, and most
preferably at least 99% pure. "Purify" or "purification" in other
embodiments means removing at least one contaminant from the
composition to be purified. In this sense, purification does not
require that the purified compound be homogenous, e.g., 100%
pure.
[0089] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers, those containing modified
residues, and non-naturally occurring amino acid polymer.
[0090] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function similarly to the naturally occurring amino
acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those amino acids that are later modified,
e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, e.g., an .alpha. carbon that is bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs may have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions
similarly to a naturally occurring amino acid.
[0091] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0092] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical or associated, e.g.,
naturally contiguous, sequences. Because of the degeneracy of the
genetic code, a large number of functionally identical nucleic
acids encode most proteins. For instance, the codons GCA, GCC, GCG
and GCU all encode the amino acid alanine. Thus, at every position
where an alanine is specified by a codon, the codon can be altered
to another of the corresponding codons described without altering
the encoded polypeptide. Such nucleic acid variations are "silent
variations," which are one species of conservatively modified
variations. Every nucleic acid sequence herein which encodes a
polypeptide also describes silent variations of the nucleic acid.
One of skill will recognize that in certain contexts each codon in
a nucleic acid (except AUG, which is ordinarily the only codon for
methionine, and TGG, which is ordinarily the only codon for
tryptophan) can be modified to yield a functionally identical
molecule. Accordingly, often silent variations of a nucleic acid
which encodes a polypeptide is implicit in a described sequence
with respect to the expression product, but not with respect to
actual probe sequences.
[0093] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention. Typically conservative substitutions for
one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D),
Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine
(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M),
Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7)
Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M)
(see, e.g., Creighton, Proteins (1984)).
[0094] Macromolecular structures such as polypeptide structures can
be described in terms of various levels of organization. For a
general discussion of this organization, see, e.g., Alberts et al.,
Molecular Biology of the Cell (3rd ed., 1994) and Cantor &
Schimmel, Biophysical Chemistry Part I: The Conformation of
Biological Macromolecules (1980). "Primary structure" refers to the
amino acid sequence of a particular peptide. "Secondary structure"
refers to locally ordered, three dimensional structures within a
polypeptide. These structures are commonly known as domains.
Domains are portions of a polypeptide that often form a compact
unit of the polypeptide and are typically 25 to approximately 500
amino acids long. Typical domains are made up of sections of lesser
organization such as stretches of (-sheet and (-helices. "Tertiary
structure" refers to the complete three dimensional structure of a
polypeptide monomer. "Quaternary structure" refers to the three
dimensional structure formed, usually by the noncovalent
association of independent tertiary units. Anisotropic terms are
also known as energy terms.
[0095] A "label" or a "detectable moiety" is a composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, chemical, or other physical means. For example,
useful labels include fluorescent dyes, electron-dense reagents,
enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin,
or haptens and proteins or other entities which can be made
detectable, e.g., by incorporating a radiolabel into the peptide or
used to detect antibodies specifically reactive with the peptide.
The radioisotope may be, for example, 3H, 14C, 32P, 35S, or 125I.
In some cases, particularly using antibodies against the proteins
of the invention, the radioisotopes are used as toxic moieties, as
described below. The labels may be incorporated into the nucleic
acids, proteins and antibodies at any position. Any method known in
the art for conjugating the antibody to the label may be employed,
including those methods described by Hunter et al., Nature, 144:945
(1962); David et al., Biochemistry, 13:1014 (1974); Pain et al., J.
Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. and
Cytochem., 30:407 (1982). The lifetime of radiolabeled peptides or
radiolabeled antibody compositions may be extended by the addition
of substances that stablize the radiolabeled peptide or antibody
and protect it from degradation. Any substance or combination of
substances that stablize the radiolabeled peptide or antibody may
be used including those substances disclosed in U.S. Pat. No.
5,961,955.
[0096] An "effector" or "effector moiety" or "effector component"
is a molecule that is bound (or linked, or conjugated), either
covalently, through a linker or a chemical bond, or noncovalently,
through ionic, van der Waals, electrostatic, or hydrogen bonds, to
an antibody. The "effector" can be a variety of molecules
including, e.g., detection moieties including radioactive
compounds, fluorescent compounds, an enzyme or substrate, tags such
as epitope tags, a toxin; activatable moieties, a chemotherapeutic
agent; a lipase; an antibiotic; or a radioisotope emitting "hard"
e.g., beta radiation.
[0097] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, e.g., recombinant cells
express genes that are not found within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all. By the term "recombinant nucleic acid" herein is meant nucleic
acid, originally formed in vitro, in general, by the manipulation
of nucleic acid, e.g., using polymerases and endonucleases, in a
form not normally found in nature. In this manner, operably linkage
of different sequences is achieved. Thus an isolated nucleic acid,
in a linear form, or an expression vector formed in vitro by
ligating DNA molecules that are not normally joined, are both
considered recombinant for the purposes of this invention. It is
understood that once a recombinant nucleic acid is made and
reintroduced into a host cell or organism, it will replicate
non-recombinantly, i.e., using the in vivo cellular machinery of
the host cell rather than in vitro manipulations; however, such
nucleic acids, once produced recombinantly, although subsequently
replicated non-recombinantly, are still considered recombinant for
the purposes of the invention. Similarly, a "recombinant protein"
is a protein made using recombinant techniques, i.e., through the
expression of a recombinant nucleic acid as depicted above.
[0098] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not normally found in the same
relationship to each other in nature. For instance, the nucleic
acid is typically recombinantly produced, having two or more
sequences, e.g., from unrelated genes arranged to make a new
functional nucleic acid, e.g., a promoter from one source and a
coding region from another source. Similarly, a heterologous
protein will often refer to two or more subsequences that are not
found in the same relationship to each other in nature (e.g., a
fusion protein).
[0099] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding
reaction that is determinative of the presence of the protein, in a
heterogeneous population of proteins and other biologics. Thus,
under designated immunoassay conditions, the specified antibodies
bind to a particular protein sequences at least two times the
background and more typically more than 10 to 100 times background.
The antibodies of the invention specifically bind to Wnt or
Frizzled proteins or other proteins in a wnt/fzd signaling pathway.
By "specifically bind" herein is meant that the antibodies bind to
the protein with a K.sub.D of at least about 0.1 mM, more usually
at least about 1 .mu.M, preferably at least about 0.1 .mu.M or
better, and most preferably, 0.01 .mu.M or better.
[0100] Specific binding to an antibody under such conditions
requires an antibody that is selected for its specificity for a
particular protein. For example, polyclonal antibodies raised to a
particular protein, polymorphic variants, alleles, orthologs, and
conservatively modified variants, or splice variants, or portions
thereof, can be selected to obtain only those polyclonal antibodies
that are specifically immunoreactive with specific Wnt or specific
Frizzled proteins, or other proteins in a wnt/fzd signaling
pathway, and not with other proteins. This selection may be
achieved by subtracting out antibodies that cross-react with other
molecules. A variety of immunoassay formats may be used to select
antibodies specifically immunoreactive with a particular protein.
For example, solid-phase ELISA immunoassays are routinely used to
select antibodies specifically immunoreactive with a protein (see,
e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for
a description of immunoassay formats and conditions that can be
used to determine specific immunoreactivity).
[0101] "Tumor cell" refers to precancerous, cancerous, and normal
cells in a tumor.
[0102] "Cancer cells," "transformed" cells or "transformation" in
tissue culture, refers to spontaneous or induced phenotypic changes
that do not necessarily involve the uptake of new genetic material.
Although transformation can arise from infection with a
transforming virus and incorporation of new genomic DNA, or uptake
of exogenous DNA, it can also arise spontaneously or following
exposure to a carcinogen, thereby mutating an endogenous gene. In
the present invention transformation is typically associated with
overexpression of Wnt and/or Frizzled proteins. Transformation is
associated with other phenotypic changes, such as immortalization
of cells, aberrant growth control, nonmorphological changes, and/or
malignancy (see, Freshney, Culture of Animal Cells a Manual of
Basic Technique (3rd ed. 1994)).
BRIEF DESCRIPTION OF THE DRAWINGS
[0103] FIG. 1. Several developmental signaling pathways are
depicted.
[0104] FIG. 2. RT-PCR analysis of a subset of HNSCC and B-cell
lines for frizzled 2 mRNA.
[0105] FIG. 3. A western blot analysis of tumor and normal cells
for frizzled 2, wnt1 and 10b.
[0106] FIGS. 4A, 4B, and 4C. An inhibition of proliferation assay
in a HNSCC line is depicted. Specifically, anti-frizzled 2,
anti-wnt 1, and anti-wnt 10b are tested for their ability to
inhibit proliferation.
[0107] FIG. 5. Apoptotic effects of inhibition of the Wnt/Frizzled
signaling pathway in a HNSCC line is depicted.
[0108] FIG. 6. Sequence alignment of a portion of the first
extracellular region of human Frizzled receptors is depicted.
[0109] FIGS. 7A and 7B. FIG. 7A depicts an immunoblot after
treatment with Wnt 1 or Wnt 10b antibodies. SNU1076 cells were
treated for 72 hrs with 2 .mu.g/ml of anti-Wnt 1, Wnt 10b, or
control antibodies. FIG. 7B shows that treatment with Wnt1
antibodies reduces transcription of TCF/LEF gene.
[0110] FIGS. 8A and 8B. FIG. 8A depicts an RT-PCR amplification for
Wnt/FZD families in cancer cell lines. FIG. 8B depicts an RT-PCR
amplification for Wnt/FZD families in normal cells.
[0111] FIGS. 9A and 9B. Protein expression of FZD 2, Wnt 1, Wnt
10b, .beta.-catenin and actin in normal and malignant cells.
[0112] FIG. 10. Inhibition of proliferation of the SNU 1076 cell
line Wnt 1 and Wnt 10b.
[0113] FIG. 11. Growth inhibition with a soluble WNT antagonist,
secreted frizzled related protein (SFRP).
[0114] FIG. 12. Apoptotic effect of inhibition of the Wnt/Frizzled
signaling pathway in a HNSCC line.
[0115] FIGS. 13A and 13B. Primer sequences for wnt, fzd, and
wnt-related gene analysis.
[0116] FIG. 14. Expression of wnt's in non-tumor and tumor
tissues.
[0117] FIG. 15. Expression of wnt 14 in non-tumor and tumor
tissues.
[0118] FIG. 16. Expression of fzd's in non-tumor and tumor
tissues.
[0119] FIG. 17. Expression of fzd3 in non-tumor and tumor
tissues.
[0120] FIG. 18. Expression of fzd6 in non-tumor and tumor
tissues.
[0121] FIG. 19. Expression of fzd10 in non-tumor and tumor
tissues.
[0122] FIG. 20. Expression of DKK's in non-tumor and tumor
tissues.
[0123] FIG. 21. Expression of FRP2/4 in non-tumor and tumor
tissues.
[0124] FIG. 22. Expression of WISP3 in non-tumor and tumor
tissues.
[0125] FIG. 23. Expression of cyclin D1 in non-tumor and tumor
tissues.
[0126] FIG. 24. Expression of c-myc in non-tumor and tumor
tissues.
[0127] FIG. 25. Expression of IL-6 in non-tumor and tumor
tissues.
[0128] FIG. 26. Expression of MMP3 in non-tumor and tumor
tissues.
[0129] FIG. 27. Expression of wnt, fzd, and wnt-related genes in
non-tumor cells and breast cancer cells.
DETAILED DESCRIPTION OF FIGURES
[0130] FIG. 1. Schematic of developmental signaling pathways is
depicted. The signalling pathways of the Wnt/wingless and
Hedehog/Sonic hedgehog are shown. Both sets of ligands interact
with a cell surface receptor. Proteins involved in the signaling
pathway are shown, for example, LEF1 and GSK3.
[0131] FIG. 2. RT-PCR analysis of a subset of HNSCC and B-cell
lines for frizzled 2 mRNA. Total RNA was extracted from HNSCC lines
(PCI13, Detroit 562, RPMI 2650, SNU1076, KB, AMC4), a CLL line
(Lesch), a Burkitt lymphoma line (Ramos), glioma lines (U87MG, and
U373MG), normal human bronchial epithelial cell lines (Clonetics,
San Diego, Calif.) and normal oral squamous epithelial (OSE) cells
using RNAzol (Gibco BRL, Grand Island, N.Y.). Reverse transcription
was performed using 1 .mu.g of RNA from each sample and the
Superscript.TM. Preamplification kit (Gibco BRL). Frizzled 2 was
amplified with 25 cycles of PCR. G3PDH mRNA was amplified in a
separate reaction for each sample.
[0132] FIG. 3. A sample western blot analysis of tumor and normal
cells for frizzled 2, wnt 5A and 10b. Adherent cells in culture
were harvested and lysed with a solution containing 25 mM Tris HCl,
150 mM KCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholic acid, 0.1%
sodium dodecyl sulfate, 1 mM NaVO.sub.3, 1 mM NaF, 20 mM
.beta.-glycerophosphate and protease inhibitors. Twenty .mu.g of
protein from each cell line was separated by SDS-PAGE and
transferred to a PVDF membrane. The membrane was immersed in 2%
I-block, 0.05% Tween X in PBS and then incubated with a 1:500
dilution of polyclonal goat anti-human Wnt 1, Wnt 10b, or frizzled
2 IgG (Santa Cruz Biotechnology, Santa Cruz, Calif.). These primary
antibodies were then detected by horseradish peroxidase-conjugated
donkey anti-goat IgG (Santa Cruz) and chemiluminescence (ECL
detection reagents, Amersham Life Science, Aylesbury, UK). To
verify relative amount of protein transferred in each lane, the
presence of actin was measured with an actin monoclonal antibody
(Chemi-Con International Inc, Temecula, Calif.).
[0133] FIGS. 4A, 4B, and 4C. Inhibition of proliferation in a HNSCC
line. Briefly, either 7.5.times.10.sup.3 or 10.times.10.sup.3
SNU1076 cells per well were seeded in 96 well plates. After 24
hours, graded amounts of polyclonal goat anti-human frizzled 2, Wnt
1, or Wnt 10b IgG (sAB)(Santa Cruz' Biotechnology, Santa Cruz,
Calif.), or control goat anti-human IgG (cAB)(Fisher Scientific,
Pittsburgh, Pa.) were added. On days 1, 2, 3, or 4, 20 .mu.l of MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
bromide)-based solution was added to wells for four hours prior to
lysis with 15% SDS, 0.015 M HCl. Absorbencies of 570 and 650 nm
were measured.
[0134] FIG. 5. Apoptotic effect of inhibition of the Wnt/Frizzled
signaling pathway in a HNSCC line. The HNSCC line SNU1076, growing
in RPMI-1640 supplemented with 10% FBS, was treated for 72 hrs with
300 ng/ml anti-Frizzled 2, Wnt-1, Wnt10b, or control nonspecific
polyclonal antibodies. The cytotoxic effects of these antibodies
were assessed by vital dye retention and DNA content. Panel A:
cells were detached from the flasks by trypsin treatment and
incubated for 10 minutes in growing medium with 5 .mu.g/ml
Propidium iodide (PI) and 40 nM DiOC.sub.6 and analyzed by flow
cytometry. Viable cells (stripes) had high DiOC.sub.6 (FL-1) and
low PI (FL-3) fluorescence, and apoptotic cells (stippled) had low
DiOC.sub.6 (FL-1) and low PI (FL-3) fluorescence. Panel B: cells
were detached from the flasks by trypsin treatment and incubated
overnight in a hypotonic buffer (0.1% citrate, 0.1% SDS) containing
50 .mu.g/ml PI and 100 .mu.g/ml RNase. The amount of DNA was then
measured by flow cytometry, and apoptotic cells were defined as
having a DNA content lower than the G.sub.0G.sub.1 levels
(sub-G.sub.0 cells).
[0135] FIG. 6. Sequence alignment of a portion of the first
extracellular region of human Frizzled receptors. Specifically, the
amino acid sequences of HFZ1 through BTZ10 are aligned to show
similarity.
[0136] FIGS. 7A and 7B. FIG. 7A: immunoblot after treatment with
Wnt 1 or Wnt 10b antibodies. SNU1076 cells were treated for 72 hrs
with 2 .mu.g/ml of anti-Wnt 1, Wnt 10b, or control antibodies.
Twenty .mu.g of protein from each cell line was separated by
SDS-PAGE and transferred to a PVDF membrane. The membrane was
immersed in 2% I-block, 0.05% Tween X in PBS and then incubated
with a monoclonal anti-human .beta.-catenin, cyclin D1, or
fibronectin IgG. These primary antibodies were then detected by
horseradish peroxidase-conjugated anti-IgG and chemiluminescence.
To verify and compare relative amounts of protein in each lane,
PVDF membrane was stripped with Re-Blot.TM. Western blot recycling
kit and reprobed for other antibodies or actin monoclonal antibody.
FIG. 7B: treatment with Wnt1 antibodies reduces transcription of
TCF/LEF gene. SNU 1076 cells were treated with 2 .mu.g/ml of
anti-Wnt-1, or control antibodies for 36 hrs. SNU 1076 cells were
cotransfected with 0.5 .mu.g/ml of pTOPFLASH-Luc or
pFOPFLASH-Luc-and 0.5 .mu.g/ml of pCMV-.beta.Gal. Cells were
harvested 24h after transfection, and lysed in lysis buffer.
Luciferase and .beta.-galactosidase activities determined using
Dual-Light.TM. reporter gene assay system. Luciferase activities of
each of pTOPFLASH-Luc or pFOPFLASH-Luc and .beta.-galactosidase
activities of pCMV-.beta.Gal were measured in the same sample by
luminometer. Transfection efficiency of each sample was normalized
by the activity of .beta.-galactosidase activity.
[0137] FIGS. 8A and 8B. FIG. 8A: RT-PCR amplification for Wnt/FZD
families in cancer cell lines. Lane 1: DNA standard, lane 2:
H.sub.2O, Lanes 3 and 4: glioblastoma, lanes 5-14: head and neck
cancers, lanes 15 and 16: B cell cancers. FIG. 8B: RT-PCR
amplification for Wnt/FZD families in normal cells. Lane 1: DNA
standard, lane 2: H.sub.2O, lanes 7 and 14: normal human bronchial
epithelial cell, other lanes: normal oral squmous cells.
[0138] FIGS. 9A and 9B. Protein expression of FZD 2, Wnt 1, Wnt
10b, .beta.-catenin and actin in normal and malignant cells. Normal
oral squamous epithelium (OSE), normal human broncheotracheal
epithelial cells (NHBE), HNSCC lines, and other solid and B cell
tumor lines were lysed, separated by SDS-page, blotted onto PDVF
membranes and successively probed with the indicated
antibodies.
[0139] FIG. 10. Inhibition of proliferation of the SNU 1076 cell
line. 7.5.times.10.sup.3 SNU 1076 cells per well were seeded in 96
well plates. After 24 hours, graded amounts of polyclonal goat
anti-human Wnt 1, Wnt 10b, or control goat anti-human IgG were
added. On days 1, 2, 3, or 4, 20 .mu.L of MTT solution was added to
wells for four hours prior to lysis with 15% SDS, 0.015 M HCl.
Absorbencies of 570 and 650 nm were measured. Data are expressed as
the mean of at least 4 independent experiments.+-.SD.
[0140] FIG. 11. Growth inhibition with a soluble WNT antagonist,
secreted frizzled related protein (SFRP). Cell viability of two
HNSCC lines was determined with MTT assay 72 hours after addition
of 2 .mu.g/ml of recombinant human SFRP 1. Data are expressed as
the mean of 2 independent experiments.+-.SD.
[0141] FIG. 12. Apoptotic effect of inhibition of the Wnt/Frizzled
signaling pathway in a HNSCC line. SNU1076 was treated for 72 hrs
with 2 .mu.g/ml of anti-Wnt 1, Wnt 10b, or control antibodies. The
cytotoxic effects of these antibodies were assessed by vital dye
retention and DNA content. Cells were detached from the flasks by
trypsin treatment and incubated for 10 minutes in growing medium
with 5 .mu.g/ml Propidium iodide (PI) and 40 nM DiOC.sub.6 and
analyzed by flow cytometry. Viable cells had high DiOC.sub.6 (FL-1)
and low PI (FL-3) fluorescence, and apoptotic cells had low
DiOC.sub.6 (FL-1) and low PI (FL-3) fluorescence.
[0142] FIGS. 13A and 13B. Primer sequences for wnt, fzd, and
wnt-related gene analysis. FIG. 13A shows primers and probes used
for analysis of wnt and fzd nucleic acid expression. FIG. 13B shows
primers and probes used for analysis of expression of Frp, WISP,
DKK, and other wnt/fzd inducible genes or controls. Levels of wnt
fzd and wnt-related genes were determined with real time PCR using
the depicted primers and probes.
[0143] FIG. 14. Expression of wnt's in non-tumor and tumor tissues.
Levels of wnt16, wnt1, wnt3, wnt7b, wnt8a, and wnt10b were
determined in normal cells from lung, colon, kidney, brain, adrenal
gland, thyroid, placenta, spleen, thymus, liver, heart, bone
marrow, and peripheral blood lymphocytes. Levels of wnt16, wnt1,
wnt3, wnt7b, wnt8a, and wnt10b were also determined in primary CLL
cells and in breast cancer tumors. The data in the figures are
relative, with the lowest normal tissue level assigned a value of
one. Thus, a relative value of 100 in breast cancer or CLL means
that the cancer cells had 100 times the values of the lowest normal
tissue, as reported by real time PCR.
[0144] FIG. 15. Expression of wnt14 in normal and non-tumor
tissues. Levels of wnt 14 were determined in normal cells from
lung, colon, kidney, brain, adrenal gland, thyroid, placenta,
spleen, thymus, liver, heart, bone marrow, and peripheral blood
lymphocytes. Levels of wnt14 were also determined in primary CLL
cells and in breast cancer tumors. Data were analyzed as in FIG.
14.
[0145] FIG. 16. Expression of fzd's in non-tumor and tumor tissues.
Levels of fzd1, fzd2, fzd3, fzd4, fzd5, fzd6, fzd7, fzd8, fzd9, and
fzd10 were determined in normal cells from lung, colon, kidney,
brain, adrenal gland, thyroid, placenta, spleen, thymus, liver,
heart, bone marrow, and peripheral blood lymphocytes. Levels of
fzd1, fzd2, fzd3, fzd4, fzd5, fzd6, fzd7, fzd8, fzd9, and fzd10
were also determined in primary CLL cells and in breast cancer
tumors. Data were analyzed as in FIG. 14.
[0146] FIG. 17. Expression of fzd3 in non-tumor and tumor tissues.
Levels of fzd3 were determined in normal cells from lung, colon,
kidney, brain, adrenal gland, thyroid, placenta, spleen, thymus,
liver, heart, bone marrow, and peripheral blood lymphocytes. Levels
of fzd3 were also determined in primary CLL cells and in breast
cancer tumors. Data were analyzed as in FIG. 14.
[0147] FIG. 18. Expression of fzd6 in non-tumor and tumor tissues.
Levels of fzd6 were determined in normal cells from lung, colon,
kidney, brain, adrenal gland, thyroid, placenta, spleen, thymus,
liver, heart, bone marrow, and peripheral blood lymphocytes. Levels
of fzd6 were also determined in primary CLL cells and in breast
cancer tumors. Data were analyzed as in FIG. 14.
[0148] FIG. 19. Expression of fzd10 in non-tumor and tumor tissues.
Levels of fzd10 were determined in normal cells from lung, colon,
kidney, brain, adrenal gland, thyroid, placenta, spleen, thymus,
liver, heart, bone marrow, and peripheral blood lymphocytes. Levels
of fzd10 were also determined in primary CLL cells and in breast
cancer tumors. Data were analyzed as in FIG. 14.
[0149] FIG. 20. Expression of DKK's in non-tumor and tumor tissues.
Levels of DKK-1, DKK-2, DKK-3, and DKK-4 were determined in normal
cells from lung, colon, kidney, brain, adrenal gland, thyroid,
placenta, spleen, thymus, liver, heart, bone marrow, and peripheral
blood lymphocytes. Levels of DKK-1, DKK-2, DKK-3, and DKK-4, were
also determined in primary CLL cells and in breast cancer tumors.
Data were analyzed as in FIG. 14.
[0150] FIG. 21. Expression of FRP2/4 in non-tumor and tumor
tissues. Levels of FRP-2 and FRP-4 were determined in normal cells
from lung, colon, kidney, brain, adrenal gland, thyroid, placenta,
spleen, thymus, liver, heart, bone marrow, and peripheral blood
lymphocytes. Levels of FRP-2 and FRP-4 were also determined in
primary CLL cells and in breast cancer tumors. Data were analyzed
as in FIG. 14.
[0151] FIG. 22. Expression of WISP3 in non-tumor and tumor tissues.
Levels of WISP3 were determined in normal cells from lung, colon,
kidney, brain, adrenal gland, thyroid, placenta, spleen, thymus,
liver, heart, bone marrow, and peripheral blood lymphocytes. Levels
of WISP3 were also determined in primary CLL cells and in breast
cancer tumors. Data were analyzed as in FIG. 14.
[0152] FIG. 23. Expression of cyclin D1 sin non-tumor and tumor
tissues. Levels of cyclin D1 were determined in normal cells from
lung, colon, kidney, brain, adrenal gland, thyroid, placenta,
spleen, thymus, liver, heart, bone marrow, and peripheral blood
lymphocytes. Levels of cyclin D1 were also determined in primary
CLL cells and in breast cancer tumors. Data were analyzed as in
FIG. 14.
[0153] FIG. 24. Expression of c-myc in non-tumor and tumor tissues.
Levels of c-myc were determined in normal cells from lung, colon,
kidney, brain, adrenal gland, thyroid, placenta, spleen, thymus,
liver, heart, bone marrow, and peripheral blood lymphocytes. Levels
of c-myc were also determined in primary CLL cells and in breast
cancer tumors. Data were analyzed as in FIG. 14.
[0154] FIG. 25. Expression of IL-6 in non-tumor and tumor tissues.
Levels of IL-6 were determined in normal cells from lung, colon,
kidney, brain, adrenal gland, thyroid, placenta, spleen, thymus,
liver, heart, bone marrow, and peripheral blood lymphocytes. Levels
of IL-6 were also determined in primary CLL cells and in breast
cancer tumors. Data were analyzed as in FIG. 14.
[0155] FIG. 26. Expression of MMP3 in non-tumor and tumor tissues.
Levels of MMP3 were determined in normal cells from lung, colon,
kidney, brain, adrenal gland, thyroid, placenta, spleen, thymus,
liver, heart, bone marrow, and peripheral blood lymphocytes. Levels
of MMP3 were also determined in primary CLL cells and in breast
cancer tumors. Data were analyzed as in FIG. 14.
[0156] FIG. 27. Expression of wnt, fzd, and wnt-related genes in
non-tumor cells and breast cancer cells. Levels of wnt, fzd, and
wnt-related genes wre determined in normal cells and breast cancer
cells. Results are expressed as fold induction. Data were analyzed
as in FIG. 14.
DETAILED DESCRIPTION OF THE INVENTION
[0157] This invention is based, at least in part, on the discovery
that particular Wnt and Frizzled proteins are associated with
different cancers. It is known that Wnt proteins often have high
level expression in cancer. However, little is known regarding the
expression of particular Wnt and Frizzled proteins. The present
disclosure provides methods for evaluating the expression of Wnt
and Frizzled proteins. Also disclosed are agents useful for
treating cancers that overexpress Wnt proteins. The invention is
useful for any cancer in which Wnt-Fzd signaling affects cancer
cell growth or survival; or in which specific wnt gene products
and/or specific fzd gene products are overexpressed. The invention
is useful for treating cancers such as head and neck cancer,
glioblastoma, chronic lymphocytic leukemia, breast cancer, mantle
zone lymphomas, Burkitt's lymphoma, and other lymphocyte
malignancies.
[0158] Applicants provide novel primers that are useful to assess
wnt and/or fzd expression levels in a system of choice. In some
embodiments wnt and/or fzd levels are determined in cell lines
derived from primary cancer cell, e.g., from a solid tumor or from
a hematopoetic cancer. In other embodiments wnt and/or fzd levels
are determined in primary tissues, e.g., solid tumors or
hematopoetic cancer cells. Cells from normal tissue or from
non-transformed cell lines are used as controls. Wnt and/or fzd
overexpression can also be determined by using antibodies against
the specific wnt or fzd protein to determine expression levels.
[0159] Overexpression of a specific wnt or fzd gene product in a
cancer cell can be based, inter alia, on two different comparisons.
First, a specific wnt and/or fzd gene product can be overexpressed
in a cancer cell relative to levels of the specific wnt and/or fzd
gene product in a noncancerous cell from the same tissue-type.
Alternatively, a specific wnt and/or fzd gene product can be
overexpressed in a cancer cell relative to levels of different
specific wnt gene product in the same cancer cell. In some
embodiments, a specific wnt and/or fzd protein is overexpressed in
a cancer cell relative to levels of the specific wnt and/or fzd
gene product in a noncancerous cell from the same tissue-type, and
relative to levels of different specific wnt gene product in the
same cancer cell.
[0160] Wnt and/or fzd overexpression can result in at least two
different outcomes for the cancer cell. In some embodiments,
expression of specific wnt and/or fzd gene products is not required
for cancer cell survival, but rather can serve as a marker of the
cancer cells. In other embodiments, expression of a specific wnt
and/or fzd will be required for proliferation or survival or for
inhibition of apoptosis of the cancer cell. (E.g., Blocking wnt/fzd
binding or wnt/fzd signaling with, for example a specific antibody
or wnt antagonist results in decreased cell proliferation or
induction of apoptosis.) Without wishing to be bound by theory,
expression of specific wnt and/or fzd gene products can lead to
activation of a signal transduction pathway and regulation of
downstream wnt and/or fzd inducible genes. In some embodiments,
activation of the signal transduction pathway and induction of the
downstream genes and gene products are required for proliferation
or survival or for inhibition of apoptosis of the cancer cell. For
example, in breast cancer cells expression of specific wnt and/or
fzd proteins appears to induce required genes including cyclin D1,
c-myc, and WISP family genes. In some cells wnt expression appears
to activate TCF/LEF transcription factors leading to induction of
specific genes. In other embodiments, activation of the signal
transduction pathway and induction of the downstream genes and gene
products are not required for proliferation or survival or for
inhibition of apoptosis of the cancer cell.
[0161] At least two therapies can be based on detection of wnt
and/or fzd overexpression. For wnt and/or fzd gene products that
are required for cell growth, survival or inhibition of apoptosis,
specific antibodies that block the wnt/fzd signaling pathway, such
as the wnt/fzd interaction, or specific antagonists can be used to
kill the cells or to induce apoptosis. For wnt and or/fzd gene
products that are overexpressed but not required for cell survival,
wnt and or fzd specific antibodies can be radiolabeled or
conjugated to toxins or can be used to induce the complement
cascade. The overexpressed wnt and/or fzd gene products act as
markers to guide the antibodies to the cancer cells. Specific
radiolabeled or toxin-conjugated antibodies or induction of the
complement cascade can also be used to assist killing of cancer
cells that overexpress specific wnts and/or fzds that are required
for cell growth.
[0162] Wnt and/or fzd expression can be correlated with the
expression of wnt/fzd induced genes (e.g., a downstream wnt/fzd
regulated gene product). For example, in breast cancer cells
expression of specific wnt and fzd proteins appears to induce
required genes including cyclin D1, c-myc, and WISP family genes.
In lymphocyte cells wnt expression appears to activate TCF/LEF
transcription factors leading to induction of specific genes. Thus,
different cancers can overexpress different wnt and fzd gene
products, as well as different downstream wnt/fzd regulated gene
products. Correlation of expression of a specifc wnt and/or fzd
gene with a specific downstream gene is an indication that the
overexpressed wnt and/or fzd gene product is active. Thus, an assay
that detects Wnt overexpression coupled with induced expression of
wnt/fzd downstream gene products provides evidence that treatment
with an agent that blocks wnt/fzd signaling is appropriate.
[0163] Antibodies to WNT and Frizzled Proteins
[0164] As noted above, the invention provides methods of inhibiting
the wnt/fzd pathway, including Wnt signaling in cancer cells. In
some embodiments of the invention, antibodies are used to block the
binding between Wnt ligand and the Frizzled receptor, or otherwise
block a step in a wnt/fzd signaling pathway. The antibodies can
also be used to induce the complement cascade against a target cell
expressing the target antigen or can be radiolabeled or
toxin-conjugated. This is particularly useful if the antigen is a
Frizzled receptor overexpressed on a target cancer cell. The
antibodies can be raised against either Wnt or Frizzled proteins,
or other proteins in the wnt/fzd pathway in some embodiments.
Alternatively, the antibodies could be raised against the
Wnt/Frizzled complex on the surface of the cell. Such antibodies
will provide more specificity by binding only cells in which the
target Wnt and Frizzled proteins are associated and can be used
without modification or can be radiolabeled or
toxin-conjugated.
[0165] Methods of preparing polyclonal antibodies are known to the
skilled artisan (e.g., Coligan, supra; and Harlow & Lane,
supra). Polyclonal antibodies can be raised in a mammal, e.g., by
one or more injections of an immunizing agent and, if desired, an
adjuvant. Typically, the immunizing agent and/or adjuvant will be
injected in the mammal by multiple subcutaneous or intraperitoneal
injections. The immunizing agent may include a protein encoded by a
nucleic acid of the figures, or fragment thereof, or a fusion
protein thereof. It may be useful to conjugate the immunizing agent
to a protein known to be immunogenic in the mammal being immunized.
Examples of such immunogenic proteins include but are not limited
to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin,
and soybean trypsin inhibitor. Examples of adjuvants which may be
employed include Freund's complete adjuvant and MPL-TDM adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The
immunization protocol may be selected by one skilled in the art
without undue experimentation.
[0166] The antibodies may, alternatively, be monoclonal antibodies.
Monoclonal antibodies may be prepared using hybridoma methods, such
as those described by Kohler & Milstein, Nature 256:495 (1975).
In a hybridoma method, a mouse, hamster, or other appropriate host
animal, is typically immunized with an immunizing agent to elicit
lymphocytes that produce or are capable of producing antibodies
that will specifically bind to the immunizing agent. Alternatively,
the lymphocytes may be immunized in vitro. Generally, either
peripheral blood lymphocytes ("PBLs") are used if cells of human
origin are desired, or spleen cells or lymph node cells are used if
non-human mammalian sources are desired. The lymphocytes are then
fused with an immortalized cell line using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell (Goding,
Monoclonal Antibodies: Principles and Practice, pp. 59-103 (1986)).
Immortalized cell lines are usually transformed mammalian cells,
particularly myeloma cells of rodent, bovine and human origin.
Usually, rat or mouse myeloma cell lines are employed. The
hybridoma cells may be cultured in a suitable culture medium that
preferably contains one or more substances that inhibit the growth
or survival of the unfused, immortalized cells. For example, if the
parental cells lack the enzyme hypoxanthine guanine phosphoribosyl
transferase (HGPRT or HPRT), the culture medium for the hybridomas
typically will include hypoxanthine, aminopterin, and thymidine
("HAT medium"), which substances prevent the growth of
HGPRT-deficient cells.
[0167] In some embodiments, a monoclonal antibody is used. The
ability of a particular antibody to recognize the same epitope as
another antibody is typically determined by the ability of one
antibody to competitively inhibit binding of the second antibody to
the antigen. Any of a number of competitive binding assays can be
used to measure competition between two antibodies to the same
antigen. For example, a sandwich ELISA assay can be used for this
purpose. This is carried out by using a capture antibody to coat
the surface of a well. A subsaturating concentration of
tagged-antigen is then added to the capture surface. This protein
will be bound to the antibody through a specific antibody:epitope
interaction. After washing a second antibody, which has been
covalently linked to a detectable moeity (e.g., HRP, with the
labeled antibody being defined as the detection antibody) is added
to the ELISA. If this antibody recognizes the same epitope as the
capture antibody it will be unable to bind to the target protein as
that particular epitope will no longer be available for binding. If
however this second antibody recognizes a different epitope on the
target protein it will be able to bind and this binding can be
detected by quantifying the level of activity (and hence antibody
bound) using a relevant substrate. The background is defined by
using a single antibody as both capture and detection antibody,
whereas the maximal signal can be established by capturing with an
antigen specific antibody and detecting with an antibody to the tag
on the antigen. By using the background and maximal signals as
references, antibodies can be assessed in a pair-wise manner to
determine epitope specificity.
[0168] A first antibody is considered to competitively inhibit
binding of a second antibody, if binding of the second antibody to
the antigen is reduced by at least 30%, usually at least about 40%,
50%, 60% or 75%, and often by at least about 90%, in the presence
of the first antibody using any of the assays described above.
[0169] In some embodiments the antibodies to the Wnt or Frizzled
proteins are chimeric or humanized antibodies. As noted above,
humanized forms of antibodies are chimeric immunoglobulins in which
residues from a complementary determining region (CDR) of human
antibody are replaced by residues from a CDR of a non-human species
such as mouse, rat or rabbit having the desired specificity,
affinity and capacity.
[0170] Human antibodies can be produced using various techniques
known in the art, including phage display libraries (Hoogenboom
& Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol.
Biol. 222:581 (1991)). The techniques of Cole et al. and Boerner et
al. are also available for the preparation of human monoclonal
antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy,
p. 77 (1985) and Boerner et al., J. Immunol. 147(1):86-95 (1991)).
Similarly, human antibodies can be made by introduction of human
immunoglobulin loci into transgenic animals, e.g., mice in which
the endogenous immunoglobulin genes have been partially or
completely inactivated. Upon challenge, human antibody production
is observed, which closely resembles that seen in humans in all
respects, including gene rearrangement, assembly, and antibody
repertoire. This approach is described, e.g., in U.S. Pat. Nos.
5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016,
and in the following scientific publications: Marks et al.,
Bio/Technology 10:779-783 (1992); Lonberg et al., Nature
368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et
al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature
Biotechnology 14:826 (1996); Lonberg & Huszar, Intern. Rev.
Immunol. 13:65-93 (1995).
[0171] In some embodiments, the antibody is a single chain Fv
(scFv). The V.sub.H and the V.sub.L regions of a scFv antibody
comprise a single chain which is folded to create an antigen
binding site similar to that found in two chain antibodies. Once
folded, noncovalent interactions stabilize the single chain
antibody. While the V.sub.H and V.sub.L regions of some antibody
embodiments can be directly joined together, one of skill will
appreciate that the regions may be separated by a peptide linker
consisting of one or more amino acids. Peptide linkers and their
use are well-known in the art. See, e.g., Huston et al., Proc.
Nat'l Acad. Sci. USA 8:5879 (1988); Bird et al., Science 242:4236
(1988); Glockshuber et al., Biochemistry 29:1362 (1990); U.S. Pat.
No. 4,946,778, U.S. Pat. No. 5,132,405 and Stemmer et al.,
Biotechniques 14:256-265 (1993). Generally the peptide linker will
have no specific biological activity other than to join the regions
or to preserve some minimum distance or other spatial relationship
between the V.sub.H and V.sub.L. However, the constituent amino
acids of the peptide linker may be selected to influence some
property of the molecule such as the folding, net charge, or
hydrophobicity. Single chain Fv (scFv) antibodies optionally
include a peptide linker of no more than 50 amino acids, generally
no more than 40 amino acids, preferably no more than 30 amino
acids, and more preferably no more than 20 amino acids in length.
In some embodiments, the peptide linker is a concatamer of the
sequence Gly-Gly-Gly-Gly-Ser, preferably 2, 3, 4, 5, or 6 such
sequences. However, it is to be appreciated that some amino acid
substitutions within the linker can be made. For example, a valine
can be substituted for a glycine.
[0172] Methods of making scFv antibodies have been described. See,
Huse et al., supra; Ward et al. supra; and Vaughan et al., supra.
In brief, mRNA from B-cells from an immunized animal is isolated
and cDNA is prepared. The cDNA is amplified using primers specific
for the variable regions of heavy and light chains of
immunoglobulins. The PCR products are purified and the nucleic acid
sequences are joined. If a linker peptide is desired, nucleic acid
sequences that encode the peptide are inserted between the heavy
and light chain nucleic acid sequences. The nucleic acid which
encodes the scFv is inserted into a vector and expressed in the
appropriate host cell. The scFv that specifically bind to the
desired antigen are typically found by panning of a phage display
library. Panning can be performed by any of several methods.
Panning can conveniently be performed using cells expressing the
desired antigen on their surface or using a solid surface coated
with the desired antigen. Conveniently, the surface can be a
magnetic bead. The unbound phage are washed off the solid surface
and the bound phage are eluted.
[0173] Regardless of the method of panning chosen, the physical
link between genotype and phenotype provided by phage display makes
it possible to test every member of a cDNA library for binding to
antigen, even with large libraries of clones.
[0174] In some embodiments, the antibodies are bispecific
antibodies. Bispecific antibodies are monoclonal, preferably human
or humanized, antibodies that have binding specificities for at
least two different antigens or that have binding specificities for
two epitopes on the same antigen. In one embodiment, one of the
binding specificities is for the Wnt or Frizzled protein, the other
one is for another cancer antigen. Alternatively, tetramer-type
technology may create multivalent reagents.
[0175] As noted above, in some embodiments, the antibody is able to
fix complement. Alternatively, the antibody is conjugated to an
effector moiety. The effector moiety can be any number of
molecules, including labeling moieties such as radioactive labels
or fluorescent labels, or can be a therapeutic moiety. If the
effector moiety is a therapeutic moiety, it will typically be a
cytotoxic agent. In this method, targeting the cytotoxic agent to
cancer cells, results in direct killing of the target cell. This
embodiment is preferably carried out using antibodies against the
Frizzled receptor. Cytotoxic agents are numerous and varied and
include, but are not limited to, cytotoxic drugs or toxins or
active fragments of such toxins. Suitable toxins and their
corresponding fragments include diphtheria A chain, exotoxin A
chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin,
enomycin, auristatin and the like. Cytotoxic agents also include
radiochemicals made by conjugating radioisotopes to antibodies
raised against Wnt or Frizzled proteins, or binding of a
radionuclide to a chelating agent that has been covalently attached
to the antibody.
[0176] Identification of Particular WNT/FZD Proteins
[0177] As noted above, the invention provides means for determining
which Wnt and/or frizzled proteins are overexpressed by a
particular cancer cell. In a preferred embodiment, the expression
of each Wnt or Frizzled protein expressed in a particular cancer
cell is compared to the corresponding expression in non-cancer
cells of the same cell type. Wnt or Frizzled proteins that are
overexpressed in cancer cells compared to that in non-cancer cells
of the same type are selected as targets. In addition, to identify
the proteins most likely responsible for cellular proliferation or
survival, the Wnt or Frizzled proteins expressed by the cancer cell
are also compared. Those proteins that are overexpressed compared
to normal cells and those that are overexpressed compared to other
Wnt or Frizzled proteins in the cancer cell are selected as
targets.
[0178] Means for detecting and measuring gene expression or protein
activity are well known in the art. Such methods include detecting
the gene transcript (e.g. mRNA), measuring the quantity of
translated protein, or measuring the gene product activity. In
another preferred embodiment, a transcript (e.g., mRNA) can be
measured using amplification (e.g. PCR) based methods as described
above for directly assessing copy number of DNA. In a preferred
embodiment, transcript level is assessed by using reverse
transcription PCR (RT-PCR). PCR primers particularly useful for
amplification of desired Wnt or Frizzled proteins are provided
below.
[0179] In another preferred embodiment, transcript level is
assessed by using real time PCR. RNA is isolated from a sample of
interest. PCR primers are designed to amplify the specific gene of
interest. PCR product accumulation is measured using a dual-labeled
flourogenic oligonucleotide probe. The probe is labeled with two
different flourescent dyes, the 5' terminus reporter dye and the 3'
terminus quenching dye. The oligonucleotide probe is selected to be
homologous to an internal target sequence present in the PCR
amplicon. When the probe is intact, energy transfer occurs between
the two flourophors, and the fluorescent emission is quenched.
During the extension phase of PCR, the probe is cleaved by 5'
nuclease activity of Taq polymerase. Therefore, the reporter is no
longer in proximity to the quencher, and the increase in emission
intensity is measured. Exemplary PCR primers and hybridization
probes for amplification of desired Wnt or Frizzled proteins or
downstream wnt/fzd regulated gene products are provided in FIGS.
13A and 13B. The primers can also be used in other methods to
amplify DNA, for example RT-PCR. This assay provides a quantitative
measure of nucleic acid.
[0180] In other embodiments, once the desired amplification
products are produced, nucleic acid hybridization techniques can be
used to detect and/or quantify the gene transcript, usually after
the products are separated on a gel. The probes used in such assays
can be full length or less than the full length of the nucleic acid
sequence encoding the protein. Shorter probes are empirically
tested for specificity. Preferably nucleic acid probes are 20 bases
or longer in length, although shorter probes can also be used.
Visualization of the hybridized portions allows the qualitative
determination of the presence or absence of mRNA.
[0181] The "activity" of a Wnt or Frizzled gene can also be
detected and/or quantified by detecting or quantifying the
expressed polypeptide. The polypeptide can be detected and
quantified by any of a number of means well known to those of skill
in the art. These may include analytic biochemical methods such as
electrophoresis, capillary electrophoresis, high performance liquid
chromatography (HPLC), thin layer chromatography (TLC),
hyperdiffusion chromatography, and the like. The isolated proteins
can also be sequenced according to standard techniques to identify
polymorphisms.
[0182] The antibodies of the invention can also be used to detect
Wnt or Frizzled proteins, or cells expressing them, using any of a
number of well recognized immunological binding assays (see, e.g.,
U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For
a review of the general immunoassays, see also Methods in Cell
Biology, Vol. 37, Asai, ed. Academic Press, Inc. New York (1993);
Basic and Clinical Immunology 7th Edition, Stites & Terr, eds.
(1991).
[0183] The methods of the invention can also be used for diagnosis.
Thus, the present invention provides methods of detecting cells
that over-express Wnt or Frizzled proteins in a patient suspected
of having a particular cancer. In one method, a biopsy is performed
on the subject and the collected tissue is tested in vitro. The
tissue or cells from the tissue is then contacted with PCR primers
disclosed here to determine the level of expression. Alternatively,
an anti-Wnt or anti-Frizzled antibody of the invention can be used.
Any immune complexes which result indicate the presence of the
target protein in the biopsied sample. To facilitate such
detection, the antibody can be radiolabeled or coupled to an
effector molecule which is a detectable label, such as a
radiolabel. In another method, the cells can be detected in vivo
using typical imaging systems. Then, the localization of the label
is determined by any of the known methods for detecting the label.
A conventional method for visualizing diagnostic imaging can be
used. For example, paramagnetic isotopes can be used for MRI.
Internalization of the antibody may be important to extend the life
within the organism beyond that provided by extracellular binding,
which will be susceptible to clearance by the extracellular
enzymatic environment coupled with circulatory clearance.
[0184] Identification of Inhibitors of WNT Signaling
[0185] Wnt or Frizzled proteins (or cells expressing them) can also
be used in drug screening assays to identify agents that inhibit a
Wnt/Fzd signaling pathway. The present invention thus provides
novel methods for screening for compositions which inhibit
cancer.
[0186] Assays for Wnt/Fzd signaling can be designed to detect
and/or quantify any part of the Wnt signaling pathway. For example
the ability of an agent to affect intracellular .beta.-catenin
levels, or to induce apoptosis, or to decrease or block cellular
proliferation in target cells can be measured. Assays suitable for
these purposes are described below.
[0187] Assays may include those designed to test binding activity
to either the Wnt ligand or to the Frizzled receptor. These assays
are particularly useful in identifying agents that modulate Wnt
activity. Virtually any agent can be tested in such an assay. Such
agents include, but are not limited to natural or synthetic
polypeptides, antibodies, natural or synthetic small organic
molecules, and the like.
[0188] As noted above, a family of secreted Frizzled-related
proteins (sFRPs) function as soluble endogenous modulators of Wnt
signaling by competing with Frizzled receptors for the binding of
secreted Wnt ligands. Thus, in some format, test agents are based
on natural ligands (e.g., Wnts ligands or sFRPs) of the Frizzled
receptor.
[0189] Any of the assays for detecting Wnt signaling are amenable
to high throughput screening. High throughput assays binding assays
and reporter gene assays are similarly well known. Thus, for
example, U.S. Pat. No. 5,559,410 discloses high throughput
screening methods for proteins, U.S. Pat. No. 5,585,639 discloses
high throughput screening methods for nucleic acid binding (i.e.,
in arrays), while U.S. Pat. Nos. 5,576,220 and 5,541,061 disclose
high throughput methods of screening for ligand/antibody
binding.
[0190] In addition, high throughput screening systems are
commercially available (see, e.g., Zymark Corp., Hopkinton, Mass.;
Air Technical Industries, Mentor, Ohio; Beckman Instruments, Inc.
Fullerton, Calif.; Precision Systems, Inc., Natick, Mass., etc.).
These systems typically automate entire procedures including all
sample and reagent pipetting, liquid dispensing, timed incubations,
and final readings of the microplate in detector(s) appropriate for
the assay. These configurable systems provide high throughput and
rapid start up as well as a high degree of flexibility and
customization. The manufacturers of such systems provide detailed
protocols for various high throughput systems. Thus, for example,
Zymark Corp. provides technical bulletins describing screening
systems for detecting the modulation of gene transcription, ligand
binding, and the like.
[0191] Other assays useful in the present invention are those
designed to test neoplastic phenotypes of cancer cells. These
assays include cell growth on soft agar; anchorage dependence;
contact inhibition and density limitation of growth; cellular
proliferation; cell death (apoptosis); cellular transformation;
growth factor or serum dependence; tumor specific marker levels;
invasiveness into Matrigel; tumor growth and metastasis in vivo;
mRNA and protein expression in cells undergoing metastasis, and
other characteristics of cancer cells.
[0192] The ability of test agents to inhibit cell growth can also
be assessed by introducing the test into an animal model of
disease, and assessing the growth of cancer cells in vivo. For
example, human tumor cells can be introduced into an
immunocompromised animal such as a "nude mouse". The test agent
(e.g., a small molecule or an antibody) is administered to the
animal and the ability of the tumor cell to form tumors--as
assessed by the number and/or size of tumors formed in the
animal--is compared to tumor growth in a control animal without the
agent.
[0193] Kits Used in Diagnostic, Research, and Therapeutic
Applications
[0194] As noted above, the invention provides evidence of the
overexpression of particular Wnt or Frizzled proteins in certain
cancers. Thus, kits can be used for the detection of the particular
nucleic acids or proteins disclosed here. In diagnostic and
research applications such kits may include any or all of the
following: assay reagents, buffers, Wnt-specific or
Frizzled-specific nucleic acids or antibodies, hybridization probes
and/or primers, and the like. A therapeutic product may include
sterile saline or another pharmaceutically acceptable emulsion and
suspension base.
[0195] In addition, the kits may include instructional materials
containing directions (i.e., protocols) for the practice of the
methods of this invention. While the instructional materials
typically comprise written or printed materials they are not
limited to such. Any medium capable of storing such instructions
and communicating them to an end user is contemplated by this
invention. Such media include, but are not limited to electronic
storage media (e.g., magnetic discs, tapes, cartridges, chips),
optical media (e.g., CD ROM), and the like. Such media may include
addresses to internet sites that provide such instructional
materials.
[0196] The present invention also provides for kits for screening
for inhibitors of Wnt signaling. Such kits can be prepared from
readily available materials and reagents. For example, such kits
can comprise one or more of the following materials: a Wnt or
Frizzled polypeptide or polynucleotide, reaction tubes, and
instructions for testing the desired Wnt signaling function (e.g.,
.beta. catenin levels).
[0197] Therapeutic Methods
[0198] Administration of Inhibitors
[0199] The agents that inhibit Wnt signaling (e.g., antibodies) can
be administered by a variety of methods including, but not limited
to parenteral (e.g., intravenous, intramuscular, intradermal,
intraperitoneal, and subcutaneous routes), topical, oral, local, or
transdermal administration. These methods can be used for
prophylactic and/or therapeutic treatment.
[0200] As noted above, inhibitors of the invention can be used to
treat cancers associated with Wnt signaling. The compositions for
administration will commonly comprise a inhibitor dissolved in a
pharmaceutically acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers can be used, e.g., buffered saline
and the like. These solutions are sterile and generally free of
undesirable matter. These compositions may be sterilized by
conventional, well known sterilization techniques. The compositions
may contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, toxicity adjusting agents and the
like, for example, sodium acetate, sodium chloride, potassium
chloride, calcium chloride, sodium lactate and the like. The
concentration of active agent in these formulations can vary
widely, and will be selected primarily based on fluid volumes,
viscosities, body weight and the like in accordance with the
particular mode of administration selected and the patient's
needs.
[0201] Thus, a typical pharmaceutical composition for intravenous
administration would be about 0.1 mg to 100 g per patient per day.
Dosages from 0.1 mg to 100 g per patient per day may be used,
particularly when the drug is administered to a secluded site and
not into the blood stream, such as into a body cavity or into a
lumen of an organ. Actual methods for preparing parenterally
administrable compositions will be known or apparent to those
skilled in the art and are described in more detail in such
publications as Remington 's Pharmaceutical Science, 15th ed., Mack
Publishing Company, Easton, Pa. (1980).
[0202] The pharmaceutical compositions can be administered in a
variety of unit dosage forms depending upon the method of
administration. For example, unit dosage forms suitable for oral
administration include, but are not limited to, powder, tablets,
pills, capsules and lozenges. It is recognized that antibodies when
administered orally, should be protected from digestion. This is
typically accomplished either by complexing the molecules with a
composition to render them resistant to acidic and enzymatic
hydrolysis, or by packaging the molecules in an appropriately
resistant carrier, such as a liposome or a protection barrier.
Means of protecting agents from digestion are well known in the
art.
[0203] The compositions containing inhibitors of the invention
(e.g., antibodies) can be administered for therapeutic or
prophylactic treatments. In therapeutic applications, compositions
are administered to a patient suffering from a disease (e.g.,
breast cancer) in an amount sufficient to cure or at least
partially arrest the disease and its complications. An amount
adequate to accomplish this is defined as a "therapeutically
effective dose." Amounts effective for this use will depend upon
the severity of the disease and the general state of the patient's
health. Single or multiple administrations of the compositions may
be administered depending on the dosage and frequency as required
and tolerated by the patient. In any event, the composition should
provide a sufficient quantity of the agents of this invention to
effectively treat the patient. An amount of an inhibitor that is
capable of preventing or slowing the development of cancer in a
patient is referred to as a "prophylactically effective dose." The
particular dose required for a prophylactic treatment will depend
upon the medical condition and history of the patient, the
particular cancer being prevented, as well as other factors such as
age, weight, gender, administration route, efficiency, etc. Such
prophylactic treatments may be used, e.g., in a patient who has
previously had cancer to prevent a recurrence of the cancer, or in
a patient who is suspected of having a significant likelihood of
developing cancer.
[0204] A "patient" for the purposes of the present invention
includes both humans and other animals, particularly mammals. Thus
the methods are applicable to both human therapy and veterinary
applications. In the preferred embodiment the patient is a mammal,
preferably a primate, and in the most preferred embodiment the
patient is human.
[0205] Other known cancer therapies can be used in combination with
the methods of the invention. For example, inhibitors of Wnt
signaling may also be used to target or sensitize the cell to other
cancer therapeutic agents such as 5FU, vinblastine, actinomycin D,
cisplatin, methotrexate, and the like. In other embodiments, the
methods of the invention can be used with radiation therapy and the
like.
[0206] In some instances the antibody belongs to a sub-type that
activates serum complement when complexed with the transmembrane
protein thereby mediating cytotoxicity or antigen-dependent
cytotoxicity (ADCC). Thus, cancer can be treated by administering
to a patient antibodies directed against Wnt or Frizzled proteins
on the surface of cancer cells. Antibody-labeling may activate a
co-toxin, localize a toxin payload, or otherwise provide means to
locally ablate cells. In these embodiments, the antibody is
conjugated to an effector moiety. The effector moiety can be any
number of molecules, including labeling moieties such as
radioactive labels or fluorescent labels, or can be a therapeutic
moiety, such as a cytotoxic agent.
[0207] Use of Wnt or Frizzled Polypeptides as Vaccines
[0208] In addition to administration of inhibitors of wnt
signalling, the Wnt or Frizzled proteins or immunogenic fragments
of them can be administered as vaccine compositions to stimulate
HTL, CTL, and antibody responses against the endogenous proteins.
Such vaccine compositions can include, e.g., lipidated peptides
(see, e.g., Vitiello, et al. (1995) J. Clin. Invest. 95:341-349),
peptide compositions encapsulated in poly(D,L-lactide-co-glycolide,
"PLG") microspheres (see, e.g., Eldridge, et al. (1991) Molec.
Immunol. 28:287-294; Alonso, et al. (1994) Vaccine 12:299-306;
Jones, et al. (1995) Vaccine 13:675-681), peptide compositions
contained in immune stimulating complexes (ISCOMS; see, e.g.,
Takahashi, et al. (1990) Nature 344:873-875; Hu, et al. (1998)
Clin. Exp. Immunol. 113:235-243), multiple antigen peptide systems
(MAPs; see, e.g., Tam (1988) Proc. Nat'l Acad. Sci. USA
85:5409-5413; Tam (1996) J. Immunol. Methods 196:17-32); viral
delivery vectors (Perkus, et al., p. 379, in Kaufmann (ed. 1996)
Concepts in Vaccine Development de Gruyter; Chakrabarti, et al.
(1986) Nature 320:535-537; Hu, et al. (1986) Nature 320:537-540;
Kieny, et al. (1986) AIDS Bio/Technology 4:790-795; Top, et al.
(1971) J. Infect. Dis. 124:148-154; Chanda, et al. (1990) Virology
175:535-547), particles of viral or synthetic origin (see, e.g.,
Kofler, et al. (1996) J. Immunol. Methods 192:25-35; Eldridge, et
al. (1993) Sem. Hematol. 30:16-24; Falo, et al. (1995) Nature Med.
7:649-653).
[0209] Vaccine compositions often include adjuvants. Many adjuvants
contain a substance designed to protect the antigen from rapid
catabolism, such as aluminum hydroxide or mineral oil, and a
stimulator of immune responses, such as lipid A, Bortadella
pertussis, or Mycobacterium tuberculosis derived proteins. Certain
adjuvants are commercially available as, e.g., Freund's Incomplete
Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,
Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.);
AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such
as aluminum hydroxide gel (alum) or aluminum phosphate; salts of
calcium, iron or zinc; an insoluble suspension of acylated
tyrosine; acylated sugars; cationically or anionically derivatized
polysaccharides; polyphosphazenes; biodegradable microspheres;
monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF,
interleukin-2, -7, -12, and other like growth factors, may also be
used as adjuvants.
[0210] Vaccines can be administered as nucleic acid compositions
wherein DNA or RNA encoding the Wnt or Frizzled polypeptides, or a
fragment thereof, is administered to a patient. See, e.g., Wolff
et. al. (1990) Science 247:1465-1468; U.S. Pat. Nos. 5,580,859;
5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO
98/04720. Examples of DNA-based delivery technologies include
"naked DNA", facilitated (bupivicaine, polymers, peptide-mediated)
delivery, cationic lipid complexes, and particle-mediated ("gene
gun") or pressure-mediated delivery (see, e.g., U.S. Pat. No.
5,922,687).
[0211] Methods for the use of genes as DNA vaccines are well known,
and include placing the desired gene or portion thereof under the
control of a regulatable promoter or a tissue-specific promoter for
expression in the patient. The gene used for DNA vaccines can
encode full-length Wnt or Frizzled protein, or may encode portions
of the proteins.
[0212] In a some embodiments, the DNA vaccines include a gene
encoding an adjuvant molecule with the DNA vaccine. Such adjuvant
molecules include cytokines that increase the immunogenic response
to the polypeptide encoded by the DNA vaccine.
[0213] For therapeutic or prophylactic immunization purposes, the
peptides of the invention can be expressed by viral or bacterial
vectors. Examples of expression vectors include attenuated viral
hosts, such as vaccinia or fowlpox. This approach involves the use
of vaccinia virus, e.g., as a vector to express nucleotide
sequences that encode Wnt or Frizzled polypeptides or polypeptide
fragments. Upon introduction into a host, the recombinant vaccinia
virus expresses the immunogenic peptide, and thereby elicits an
immune response. Vaccinia vectors and methods useful in
immunization protocols are described in, e.g., U.S. Pat. No.
4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG
vectors are described in Stover, et al. (1991) Nature 351:456-460.
A wide variety of other vectors useful for therapeutic
administration or immunization e.g., adeno and adeno-associated
virus vectors, retroviral vectors, Salmonella typhi vectors,
detoxified anthrax toxin vectors, and the like, will be apparent.
See, e.g., Shata, et al. (2000) Mol. Med. Today 6:66-71; Shedlock,
et al. (2000) J. Leukoc. Biol. 68:793-806; and Hipp, et al. (2000)
In Vivo 14:571-85.
EXAMPLES
[0214] Different clonal populations of HNSCC overexpress various
receptors of the Wnt and Fzd family because of their immature cell
of origin and because of a growth and survival advantage provided
by autocrine or paracrine Wnt/Fzd signaling. We examined HNSCC and
normal human epithelial cell lines for the expression of 5 Wnt and
2 Fzd genes. The results showed that most HNSCCs did overexpress
one or more Wnt and Fzd mRNAs. Moreover, the Wnt/Fzd pathway was
functional in some of the HNSCC cells, as indicated by the
constitutive expression of a LEF/TCF reporter gene. In the SNU 1076
cell line, anti-Wnt-1 or anti-Wnt-10b antibodies decreased the
expression of .beta.-catenin and cyclin D1, inhibited cell growth,
and induced apoptosis. Thus, the Wnt and Fzd genes are frequently
overexpressed in HNSCC, and are attractive targets for both
immunotherapy and drug therapy.
[0215] We have examined tumor and normal cell lines for proteins
that are involved in embryonic development. These studies suggest
that at least one G-coupled protein receptor, frizzled 2, is
overexpressed by many tumor cell lines. A broader panel of normal
and malignant cells can be studied and immunization strategies can
be developed directed towards passive and active immunotherapies
against this antigen.
[0216] Based on the successful experience of trastuzumab as an
adjunctive passive immunotherapy as described above, an evaluation
of blocking the Wnt-frizzled signaling pathway on the growth of a
HNSCC line with commercially available polyclonal antibodies was
performed (FIGS. 4 and 5). Soluble inhibitors of frizzled have been
described to induce apoptosis secondary to their inhibition of
frizzled signaling (Zhou, Z. J. et al., "Up-regulation of human
secreted frizzled homolog in apoptosis and its down-regulation in
breast tumors," Int J Cancer 78:95-99 (1998)). The antibodies
tested appear to have slowed the growth of the tumor line and
resulted in apoptosis (FIGS. 4 and 5).
[0217] To evaluate Wnt and Fzd receptors as potential tumor
associated antigens in head and neck squamous cell cancers (HNSCC),
we screened various tumor and normal cell lines by both RT-PCR, and
immunoblotting. Initial screening revealed that both frizzled 2 and
frizzled 5 are expressed in head and neck squamous cell cancers
(HNSCC), glioma, and chronic lymphocytic leukemia (CLL) (FIG. 2).
Further, the results revealed that Fzd-2 was overexpressed in many
HNSCC cells, compared to normal human bronchoepithelial (NHBE)
cells (Table 1). The amino acid sequence of Fzd-2 is very
homologous to Fzd-1 and 7 (Sagara, N. et al. "Molecular cloning,
differential expression, and chromosomal localization of human
frizzled-1, frizzled-2, and frizzled-7," Biochem Biophys Res Comm
252, 117-122 (1998)). To confirm that frizzled 2 was specifically
amplified in the tumor
[0218] lines to RT-PCR products from selected reactions were cloned
into the TA vector (Invitrogen, Carlsbad, Calif.) and sequenced.
There was 100% identity of the inserts with the human frizzled 2
sequence by BLAST search. In addition, immunoblotting showed a lack
of detectable Fzd-2 protein in the lysates of NHBE in which there
were weakly detectable or undetectable products by RT-PCR. The
human Fzd-2 gene originally was isolated by Sagara and colleagues
(Sagara 1998, infra). These investigators also found that the mRNA
for Fzd-2 was not detectable in any of 15 different normal human
adult tissues, with the possible exception of heart. In contrast,
embryonic tissues, as well as six of eight malignant cell lines,
expressed abundant Fzd-2 mRNA. However, these investigators did not
test for the expression of frizzled Fzd-2 protein, and mRNA levels
do not necessarily correlate with protein expression. Our studies
show that Fzd-2 protein expression is prominent in HNSCC cell
lines, when compared to normal NHBE cells. Hence, antibodies
against specific determinants of the extracellular domain of Fzd-2
could be used to bind to and target such malignant cells.
[0219] Compared to NHBE cells, the HNSCC cell lines expressed much
higher message levels of Wnt-1, Wnt-5a, Wnt-10b and Wnt-13. Of
these Wnt proteins Wnt-1, 5A, and 10b were exclusively expressed by
the malignant cell lines and were not detected in the normal
tissues tested. Immunoblotting experiments confirmed the
overexpression of Wnt-1 and Wnt-10b protein in several HNSCC cell
lines (FIG. 3). Since the tumors had high levels of both the
ligands and their Fzd-2 receptors, it was important to determine if
Wnt/Fzd signaling was constitutively active in the HNSCC cells. The
canonical Wnt/Fzd signaling cascade leads to the accumulation of
cytoplasmic .beta.-catenin and its translocation to the nucleus. In
the nucleus beta-catenin binds a specific sequence motif at the N
terminus of lymphoid-enhancing factor/T cell factor (LEF/TCF) to
generate a transcriptionally active complex (Behrens J et al.
"Functional interaction of beta-catenin with the transcription
factor LEF-1," Nature 382, 638-642 (1996)). Experiments using
LEF/TCF reporter gene, TOPFLASH, demonstrated that LEF/TCF
dependent transcription was active in the SNU 1076 cells.
[0220] The Wnt/frizzled pathway has been previously implicated in
tumorigenesis. Soluble Wnt glycoproteins have been demonstrated to
transmit signal by binding to the seven transmembrane domain
G-protein coupled-receptor frizzled (FIG. 1) (Bhanot, P. et al. "A
new member of the frizzled family from Drosophila functions as a
Wingless receptor," Nature 382:225-230 (1996); Yang-Snyder, J. et
al. "A frizzled homolog functions in a vertebrate Wnt signaling
pathway," Curr Biol 6:1302-1306 (1996); Leethanakul, C. et al.
"Distinct pattern of expression of differentiation and
growth-related genes in squamous cell carcinomas of the head and
neck revealed by the use of laser capture microdissection and cDNA
arrays," Oncogene 19:3220-3224 (2000)). Upon Wnt signaling, a
cascade is initiated that results in the accumulation of
cytoplasmic beta-catenin and its translocation to the nucleus. In
the nucleus beta-catenin binds a specific sequence motif at the N
terminus of lymphoid-enhancing factor/T cell factor (LEF/TCF) to
generate a transcriptionally active complex (Behrens, J. et al.
"Functional interaction of beta-catenin with the transcription
factor LEF-1," Nature 382:638-642 (1996)). Beta-catenin interacts
with multiple other proteins such as cadherin, which it links to
the cytoskeleton (Hoschuetzky, H. et al. "Beta-catenin mediates the
interaction of the cadherin-catenin complex with epidermal growth
factor receptor," J Cell Biol 127:1375-1380 (1994); Aberle, H. et
al., "Assembly of the cadherin-catenin complex in vitro with
recombinant proteins," J Cell Sci 107:3655-3663 (1994)). It also
associates with the adenomatous polyposis coli (APC) tumor
suppressor protein and glycogen synthetase 3 beta (GSK3.beta.)
(Rubinfeld, B. et al., "Binding of GSK3beta to the APC-beta-catenin
complex and regulation of complex assembly," Science 272:1023-1026
(1996)). These proteins function to negatively regulate beta
catenin by facilitating phosphorylation near the aminoterminus and
thus accelerating its proteolytic degradation (Yost, C. et al, "The
axis-inducing activity, stability, and subcellular distribution of
beta-catenin is regulated in Xenopus embryos by glycogen synthase
kinase 3," Genes Dev 10:1443-1454 (1996)).
[0221] A panel of tumor cells that can be screened are derived from
the panel of 60 lines which are being characterized in the National
Institutes of Health Developmental Therapeutics Program. The cell
lines that are currently available include: (Non-Small Cell Lung
Cancer) A549/ATCC, NCI-11226, NCI-11460, HOP-62, HOP-92, (colon
cancer) HT29, HCT-116, (breast cancer) MCF7, NCI/ADR-RES,
MDA-MB-231/ATCC, T-47D, (ovarian cancer) OVCAR-3, OVCAR-4, SK-OV-3,
(leukemia) CCRF-CEM, K-562, MOLT-4, HL-60 (TB), RPMI-8226, (renal
cell) 786-0, TK-10, (prostate cancer) PC-3, DU-145. Normal control
cell lines can be purchased from Clonetics.
[0222] Although Wnt and Fzd were expressed in HNSCC cells, they may
be dispensable for cell growth and survival. Therefore, the effects
of antibodies to the extracellular domains of Wnt-1 and Wnt-10b
were studied in three HNSCC lines known to express the receptors.
When compared to control antibodies, both anti-Wnt antibodies
slowed the growth of one of the HNSCC cell lines (SNU 1076) and
resulted in apoptosis. Treatment with high levels of SFRP1, a Wnt
antagonist, exerted a similar effect. Moreover, interference with
Wnt/frizzled signaling in SNU 1076 cells decreased the activity of
the LEF/TCF reporter gene, and reduced levels of .beta.-catenin
cyclin D1 and fibronectin. These results suggest that continued
autocrine or paracrine Wnt/Fzd signaling may be required for the
growth and survival of a subset of HNSCC cells.
[0223] These results suggest that antibodies against Wnt and
frizzled receptors may exert two different effects in HNSCC cancers
in vivo. In malignant cells that depend on Wnt/Fzd signaling for
survival, the antibodies might directly slow tumor growth and/or
induce apoptosis. In HNSCC cells that incidentally overexpress the
receptors, but do not require them for proliferation, the
antibodies still could potentially target the tumor cells for
killing by complement, or antibody dependent cellular toxicity.
Based on these data, we believe that passive immunotherapy could be
a useful adjunctive therapy in HNSCC that overexpress one or more
Wnt and Fzd receptors.
[0224] Experimental Methods
[0225] Cell lines and culture: Ten HNSCC, 2 B lymphoma, and 2
glioblastoma cell lines were studied. Detroit-562 (pharyngeal
cancer), KB (carcinoma in the floor of the mouth), RPMI-2650 (nasal
septal) cancer), SCC-25 (tongue cancer), U87MG and U373MG
(glioblastoma), Ramos (lymphoma), Detroit-551 (human skin
fibroblast-like cells) and WI-38 (human lung fibroblasts) were
purchased from the American Type Culture Collection (Manassas,
Va.). The PCI-1, 13, and 50 cell lines were kindly provided by Dr.
T. Whiteside (Univ. of Pittsburgh, PA) (Whiteside, T. L. et al.,
"Human tumor antigen-specific T lymphocytes and
interleukin-2-activated natural killer cells: comparisons of
antitumor effects in vitro and in vivo," Clin Cancer Res. 4,
1135-1145 (1998); Yasumura, S. et al., "Human cytotoxic T-cell
lines with restricted specificity for squamous cell carcinoma of
the head and neck," Cancer Res. 53, 1461-1468 (1993)). The HNSCC
cell lines SNU 1066, SNU 1076 and AMC 4 cell lines were provided by
Dr. J. G. Park (Seoul National University, Korea) and Dr. S. Y. Kim
(University of Ulsan, Korea), respectively (Ku, J. L. et al.,
"Establishment and characterization of human laryngeal squamous
cell carcinoma cell lines," Laryngoscope 109, 976-82 (1999); Kim,
S. Y. et al. "Establishment and characterization of nine new head
and neck cancer cell lines," Acta Otolaryngol. 117, 775-784
(1997)). Two different normal human tracheobronchial epithelial
(NHBE) cells derived from different persons were purchased from
Clonetics (San Diego, Calif.). All cancer cell lines were cultured
at 37.degree. C. in a humidified atmosphere of 5% CO.sub.2, in
either RPMI 1640, DMEM (Dulbecco's modified Eagle's medium), or
Ham's 12-DMEM medium, as recommended by the suppliers, supplemented
with 10% fetal bovine serum. NHBE cells were cultured in the
bronchial epithelial cell growth media provided by the company.
Normal epithelial cells were obtained from scrapings of the oral
mucosa of 10 normal healthy volunteers. All cell lines were found
to be free of mycoplasma contamination.
[0226] RT-PCR Analyses: Total RNA was extracted by using
Trizol.RTM. (Gibco BRL, Grand Island, N.Y.), according to the
manufacturer's directions. Different pairs of gene-specific primers
based on GenBank sequences of cloned human Wnt and Fzd genes were
used for reverse transcriptase-PCR (RT-PCR) analysis. Reverse
transcription was performed with a Superscript.TM. Preamplification
kit (Gibco BRL). One microgram of RNA was used from each sample,
and 25-35 cycles of PCR were carried out. The PCR products were
separated by electrophoresis, visualized under ultra violet light,
and scanned with a laser densitometer. The intensities of the Wnt
and Fzd bands were compared with the amplicon of the housekeeping
gene G3PDH. Preliminary experiments confirmed that the PCR
amplifications had not reached a plateau for all data reported in
the results. The following list summarizes the primer pairs
used:
[0227] Fzd-2: 5'-cagcgtcttgcccgaccagatcca-3'(reverse);
5'-ctagcgccgctcttcgtgtacctg-3' (forward). Fzd-5:
5'-ttcatgtgcctggtggtgggc- -3' (forward);
5'-tacacgtgcgacagggacacc-3' (reverse).
Wnt-1:-5'-cacgacctcgtctacttcgac-3' (forward);
5'-acagacactcgtgcagtacgc-3' (reverse). Wnt-5a:
5'-acacctctttccaaacaggcc-3' (forward); 5'-ggattgttaaactcaactctc-3'
(reverse) Wnt-7a: 5'-cgcaacaagcggcccaccttc-3' (forward),
5'-tccgtgcgctcgctgcacgtg-3'(reverse) Wnt-10b:
5'-gaatgcgaatccacaacaacag; 3' (forward);
5'-ttgcggttgtgggtatcaatgaa-3'(re- verse). Wnt-13:
5'-aagatggtgccaacttcaccg-3' (forward);
5'-ctgccttcttgggggctttgc-T(reverse) G3PDH:
5'-accacagtccatgccatcaC-3' (forward);
5'-tacagcaacagggtggtggA-3'(reverse).
[0228] The specificities of the Wnt and Fzd PCR products were
confirmed by cloning and sequencing the products, using a TOPO TA
Cloning kit and M13 primers (Invitrogen, Carlsbad, Calif.).
[0229] Immunoblotting: After removal of medium, cells in
logarithmic growth were disrupted in lysis buffer [25 mM Tris HCl,
150 mM KCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholic acid, 0.1%
sodium dodecyl sulfate] including phosphatase and protease
inhibitor cocktails. Each lane of an SDS-PAGE gel was loaded with
20 .mu.g-of protein. After electrophoresis, the proteins were
transferred to a polyvinylidene difluoride (PVDF) membrane, blocked
with 2% I-block.TM. (Tropix Inc, Bedford, Mass.) containing 0.05%
Tween-X in PBS, and then incubated with primary antibody.
Horseradish peroxidase-conjugated anti-IgG (Santa Cruz
Laboratories, Santa Cruz, Calif.) was used as the secondary
antibody. The membranes were developed using a chemiluminescence
system (ECL detection reagent: Amersham Life Science, Aylesbury,
UK), and scanned with a laser densitometer. The membranes were
stripped with Re-Blot.TM. Western blot recycling kit (Chemi-Con
International Inc, Temecula, Calif.) and reprobed using other
antibodies and actin monoclonal antibody (Chemi-Con International
Inc) as a control. Prestained molecular weight markers (New England
Biolabs, Beverly, Mass.) were used as reference.
[0230] Antibodies: Polyclonal antibodies specific for the amino
terminal extracellular domains of Wnt-1 and Wnt-10b, and for the
carboxy terminal region of Fzd-2, were purchased from Santa Cruz
Laboratories, and monoclonal antibodies specific for .beta.-catenin
and fibronectin were purchased from Transduction Laboratories
(Lexington, Ky.). Antibodies to cyclin D1 and actin were purchased
from PharMingen (San Diego, Calif.) and Chemi-Con International
Inc., respectively. Purified recombinant human soluble
frizzled-related protein-1 was prepared in Dr. J. Rubin's
laboratory as described previously (Uren, A. et al., "Secreted
frizzled-related protein-i binds directly to Wingless and is a
biphasic modulator of Wnt signaling," J Biol Chem. 275, 4374-4382
(2000)).
[0231] MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
bromide)-based cell assay: Cell proliferation was determined by a
colorimetric MTT assay. Briefly, either 7.5-10.times.10.sup.3 cells
were dispersed in each well of a 96 well plate. Twenty-hours after
culture, 4 different concentrations of anti-Wnt-1 or ant-Wnt-10b
antibody (2 .mu.g/ml, 0.2 .mu.g/ml, 20 ng/ml, and 2 ng/ml) were
added to the cultures. The same concentrations of goat antihuman
IgG (Fisher Scientific) were used as an isotype control. The
antibodies were dialyzed against tissue culture medium prior to
use, to remove preservatives. On 1, 2, 3, or 4 days after
incubation, 20 .mu.l of MTT solution was added to each well. Four
hours later the cells were lysed, and absorbances at 570 nM and 650
nM were measured and growth, as a percentage of control, was
determined from the formula:
% of control growth=(B-A)/(C-A).times.100
[0232] where A=absorbance at start of incubation, B=absorbance
after incubation with antibodies tested, C=absorbance after
incubation with control antibody. The assays were performed in
triplicate, and the results represent the mean value.+-.standard
deviation from four independent experiments.
[0233] Flow Cytometry: Cell apoptosis was assayed by propidium
iodide (PI) and DIOC.sub.6 staining, followed by flow cytometry.
The HNSCC line, SNU1076, was treated with 2 .mu.g/ml anti-Wnt-1,
anti-Wnt-10, or control IgG for 72 hrs. Cells were detached from
the flasks by trypsin treatment and incubated for 10 minutes in
medium with 5 .mu.g/ml PI and 40 nM DiOC.sub.6, and then were
analyzed by flow cytometry in a FACS caliber (Becton-Dickinson, San
Jose, Calif.). Viable cells had high DiOC.sub.6 (FL-1) and low PI
(FL-3) fluorescence, whereas apoptotic cells had low DiOC.sub.6
(FL-1) and low PI (FL-3) fluorescence.
[0234] Tumor and normal cell lines can be identified that express
frizzled 2. Ten cell lines that express frizzled 2 and at least two
cell lines that do not can be tested as described above for FIG. 4.
The mouse sera that tests for highest titer and specificity will be
used in the cell cultures. The cells will be exposed to graded
amounts of polyclonal anti-frizzled 2 mouse sera and normal control
serum. On days 1, 2, 3, and 4 subsets of the replicate wells will
be assayed for proliferative capacity. On successive days 20 .mu.l
of MTT (3-[4,5-dimethylthiazol-2-yl- ]-2,5-diphenyl tetrazolium
bromide)-based solution will be added to wells for four hours prior
to lysis with 15% SDS, 0.015 M HCl. Absorbencies of 570 and 650 nm
will measured. These measurements will be performed in triplicate
and statistical relevance will be assessed by Students t test for
P<0.05.
[0235] The selected cell lines will also undergo analysis for DNA
content by Propidium iodide (PI) staining. Cell lines treated for
72 hours in the presence of graded concentrations of normal or
immunized mouse serum will be trypsinized, incubated for 10 minutes
with 5 .mu.g/ml PI and 40 nM DiOC.sub.6, and analyzed by flow
cytometry. Viable cells will be DiOC.sub.6 (FL-1) high and PI
(FL-3) low, and apoptotic cells will be DiOC.sub.6 (FL-1) low and
PI (FL-3) low. Additionally, cells will detached from the flasks
with trypsin and incubated overnight in a hypotonic buffer (0.1%
citrate, 0.1% SDS) containing 50 .mu.g/ml PI and 100 .mu.g/ml
RNase. The amount of DNA will be measured by flow cytometry.
Apoptotic cells are defined as having a DNA content lower than the
G.sub.0G.sub.1 levels (sub-G.sub.0 cells).
[0236] Transient Luciferase Assays: The pTOPFLASH-Luc reporter gene
vector and the pFOPFLASH-Luc control were kindly provided by Dr.
Hans Clevers (University Medical Center Utrecht, The Netherlands).
For TOPFLASH/FOPFLASH reporter gene assays, SNU 1076 cells were
cotransfected with 0.5 .mu.g of pTOPFLASH-Luc or pFOPFLASH-Luc and
0.5 .mu.g of pCMV-.beta.Gal, as described previously (Korinek, V.
et al., "Constitutive transcriptional activation by a
beta-catenin-Tcf complex in APC -/- colon carcinoma," Science 275,
1784-1787 (1997)). Cells were harvested 24h after transfection,
disrupted in lysis buffer, and luciferase and .beta.-galactosidase
activities were determined using the Dual-Light reporter gene assay
system (Applied Biosystems, Foster City, Calif.). Luciferase
activities of each pTOPFLASH-Luc or pFOPFLASH-Luc transfected
culture, and the .beta.-galactosidase activities of pCMV-.beta.Gal
transfected cells, were measured in the same samples using a
luminometer. The transfection efficiencies of the samples were
normalized by the activity of .beta.-galactosidase.
Example 1
Immunogenicity of Isolated Non-Homologous Regions of Frizzled 2
[0237] The first extracellular domain of frizzled 2 contains a
region which based on protein structure is least homologous to the
other frizzled protein family members (FIG. 6) (Sagara, N. et al.
"Molecular cloning, differential expression, and chromosomal
localization of human frizzled-1, frizzled-2, and frizzled-7,"
Biochem Biophys Res Commun 252:117-122 (1998)). This polypeptide
sequence may have sufficient ternary structure to generate an
antibody response to the native protein. In order to enhance B cell
stimulation this epitope will be coupled to T cell epitopes that
have been described to generate T cell help.
[0238] The overall strategy will be to use the least conserved
region of the frizzled protein, attempting to preserve the most
native structure possible and to generate the most potent immune
response. The most versatile method for designing vaccines of
defined regions is naked plasmid DNA. The advantages are that the
vectors can be rapidly redesigned to change the length of sequence
that is expressed, discontinuous regions of the protein can be
co-expressed, and the DNA sequence of the protein can be fused to
other epitopes to enhance antigenicity (O'Hem, P. A. et al.
"Colinear synthesis of an antigen-specific B-cell epitope with a
`promiscuous` tetanus toxin T-cell epitope: a synthetic peptide
immunocontraceptive," Vaccine 15:1761-1766 (1997); Paterson, M. et
al., "Design and evaluation of a ZP3 peptide vaccine in a
homologous primate model," Mol Hum Reprod 5:342-352 (1999);
Dakappagari, N. K. et al., "Prevention of mammary tumors with a
chimeric HER-2 B-cell epitope peptide vaccine," Cancer Res
60:3782-3789 (2000)). It affords the versatility of expressing
soluble, membrane bound proteins, or small peptide fragments. Also
gene transfer by this technique is a powerful tool to introduce
multiple protein elements into the same or separate locations. In
this system single or multiple proteins can be locally expressed.
Injecting a combination of plasmids expressing antigens and
costimulators like B7.1 and B7.2 results in enhanced immune
responses (Corr, M. et al., "Costimulation provided by DNA
immunization enhances antitumor immunity," J Immunol 159:4999-5004
(1997); Chan, K. et al., "The roles of mhc class ii, cd40, and b7
costimulation in ctl induction by plasmid dna (DNA?)," J Immunol
166:3061-3066 (2001)).
[0239] Several plasmids have been constructed which are under the
control of the cytomegalovirus (CMV) promoter which has been found
to enable high levels of antigen expression in injected muscle. The
pCMVint vector includes the cytomegalovirus (CMV) E1 promoter, the
simian virus (SV40) t-intron, and the SV-40 polyadenylation site
(Corr, M. et al. "Gene vaccination with naked plasmid DNA:
mechanism of CTL priming," J Exp Med 184:1555-1560 (1996)). The ACB
vector has the same elements except the polyadenylation sequence is
from the bovine growth hormone gene (Sato, Y. et al
"Immunostimulatory DNA sequences necessary for effective
intradermal gene immunization," Science 273:352-354 (1996)). The
first set of plasmid constructs planned will encode the least
homologous region of the frizzled 2 between the ninth and tenth
cysteines. These cysteines will be preserved in this series of
constructs as they may stabilize a configuration that enables
antibody binding to the native protein. This polypeptide fragment
will be fused at the aminoterminus or the carboxylterminus via a
short linker to a tetanus toxin or measles virus fusion (MVF)
protein T helper epitopes (see below) (O'Hem, P. A. et al.
"Colinear synthesis of an antigen-specific B-cell epitope with a
`promiscuous` tetanus toxin T-cell epitope: a synthetic peptide
immunocontraceptive," Vaccine 15:1761-1766 (1997); Paterson, M. et
al "Design and evaluation of a ZP3 peptide vaccine in a homologous
primate model," Mol Hum Reprod 5:342-352 (1999); Dakappagari, N. K.
et al., "Prevention of mammary tumors with a chimeric HER-2 B-cell
epitope peptide vaccine," Cancer Res 60:3782-3789 (2000)). These
minigenes will be constructed with overlapping oligonucleotides.
The oligonucleotides are 5' prime phosphorylated with T4 kinase at
room temperature for 30 minutes, annealed by boiling an equimolar
admixture of two complementary oligomers and slow cooling. The
double stranded oligonucleotides are then ligated 3' to the tissue
plasminogen leader (TPA) leader into the EcoR47111 site in frame
and into the BamHl site of the pBluescript SKII vector. The
minigene is then subcloned into the pCMV and pACB vectors between
the Pstl and Xbal sites as previously described (Corr, M. et al.,
"Costimulation provided by DNA immunization enhances antitumor
immunity," J Immunol 159:4999-5004 (1997)).
[0240] The inserts for the vectors are designed as described above.
The frizzled putative B cell epitope is from the published
sequence. The tetanus toxin and measles MVF T helper epitopes have
been optimized for human codon usage by the most frequently used
codon per amino acid. The DNA constructs have an initiating
methionine and stop codons added to the 5' and 3' ends
respectively. The aminoacid and DNA sequences are summarized below
with the short GPSL linker sequence in bold and the T cell helper
epitope underlined.
[0241] Tetanus Toxin Epitope Fused to a Frizzled Domain
1 pFZD2-TT MCVGQNHSEDGAPALLTTAPPPGLQPGAGGTPGGPGGGGAPPRYATLEHPFHC
-GPSL- VDDALINSTKIYSYFPSV-STOP ATG TGC GTC GGC CAG AAC CAC TCC GAG
GAC GGA GCT CCC GCG CTA CTC ACC ACC GCG CCG CCG CCG GGA CTG CAG CCG
GGT GCC GGG GGC ACC CCG GGT GGC CCG GGC GGC GGC GGC GCT CCC CCG CGC
TAC GCC ACG CTG GAG CAC CCC TTC CAC TGC-GGC CCC AGC CTG- GTG GAC
GAC GCC CTG ATC AAC AGC ACC AAG ATC TAC AGC TAC TTT CCC AGC GTG TAG
pTT-FZD2 MVDDALINSTKIYSYFPSV-GPSL-
CVGQNHSEDGAPALLTTAPPPGLQPGAGGTPGGPGGGGAP- PRYATLEHPFHC-STOP ATG GTG
GAC GAC GCC CTG ATC AAC AGC ACC AAG ATC TAC AGC TAC TTT CCC AGC
GTG-GGC CCC AGC CTG-TGC GTC GGC CAG AAC CAC TCC GAG GAC GGA GCT CCC
GCG CTA CTC ACC ACC GCG CCG CCG CCG GGA CTG CAG CCG GGT GCC GGG GGC
ACC CCG GGT GGC CCG GGC GGC GGC GGC GCT CCC CCG CGC TAC GCC ACG CTG
GAG CAC CCC TTC CAC TGC TAG
[0242] Measles MVF Epitope Fused to a Frizzled Domain
2 PFZD2-MMVF MCVGQNHSEDGAPALLTTAPPPGLQPGAGGTPGGPGGGGAPPRYAT-
LEHPFHC-GPSL- KLLSLIKGVIVHRLEGVE-STOP ATG TGC GTC GGC CAG AAC CAC
TCC GAG GAC GGA GCT CCC GCG CTA CTC ACC ACC GCG CCG CCG CCG GGA CTG
CAG CCG GGT GCC GGG GGC ACC CCG GGT GGC CCG GGC GGC GGC GGC GCT CCC
CCG CGC TAC GCC ACG CTG GAG CAC CCC TTC CAC TGC-GGC CCC AGC CTG-
AAG CTG CTG AGC CTG ATC AAG GGC GTG ATC GTG CAC CGC CTG GAG GGC GTG
GAG TAG PMMVF-FZD2 MKLLSLIKGVIVHRLEGVE-GPSL-
CVGQNHSEDGAPALLTTAPPPGLQPGAGGTPGGPGGGGAP- PRYATLEHPFHC-STOP ATG AAG
CTG CTG AGC CTG ATC AAG GGC GTG ATC GTG CAC CGC CTG GAG GGC GTG
GAG-GGC CCC AGC CTG-TGC GTC GGC CAG AAC CAC TCC GAG GAC GGA GCT CCC
GCG CTA CTC ACC ACC GCG CCG CCG CCG GGA CTG CAG CCG GGT GCC GGG GGC
ACC CCG GGT GGC CCG GGC GGC GGC GGC GCT CCC CCG CGC TAC GCC ACG.CTG
GAG CAC CCC TTC CAC TGC TAG
[0243] Plasmid DNA is prepared using Qiagen Maxiprep (Chatsworth,
Calif.) kits with the modification of adding one tenth volume 10%
Triton X-114 (Sigma, St. Louis, Mo.) to the clarified bacterial
lysate prior to applying it to a column. Prior to injection the
residual endotoxin level is quantified using a limulus extract clot
assay (Associates of Cape Cod, Woods Hole, Mass.). A level of less
than or equal to 5 ng endotoxin/.mu.g DNA need be obtained prior to
use in an animal (Corr, M. et al. "In vivo priming by DNA injection
occurs predominantly by antigen transfer," J Immunol 163:4721-4727
(1999)). The DNA is resuspended in a sterile pyrogen free saline
solution for injection.
[0244] Twenty-eight female mice will be divided into groups of 4
mice each. They will be injected in the dermis of the tail with a
combination of 50 pg plasmid encoding a costimulator (B7-1 or B7-2)
and 50 .mu.g linker plasmid diluted in normal saline at weeks zero,
one and two. A group with empty vector is included as a negative
control. The groups are as follows:
3 Group Plasmid 1 Plasmid 2 A pTT-FZD2 nCMV B pTT-FZD2 nCMVB7-1 C
pTT-FZD2 nCMVB7-2 D pFZD2-TT nCMV E pFZD2-TT nCMVB7-1 F pFZD2-TT
nCMVB7-2 G -- nCMV
[0245] Another group of mice in similar groups will be immunized
using the pMMVF-FZD2 and pFZD2-MMVF set of linked epitope plasmids.
The nCMVB7-1 and nCMVB7-2 constructs encode the cDNAs for murine
CD80 and CD86, which were kindly provided by G. Freeman
(Dana-Farber Cancer Institute, Boston, Mass.) (Corr, M. et al.,
"Costimulation provided by DNA immunization enhances antitumor
immunity," J Immunol 159:4999-5004 (1997)).
[0246] Mice will be bled prior to the start of the experiment and
then every two weeks thereafter. Serum will be separated and stored
at -20.degree. C. prior to testing. On week ten (seven weeks after
the last injection) mice will be sacrificed. The titers of antibody
will be tested by anti-peptide ELISA. Ninety-six well plates
(Costar) are coated with 50 .mu.l/well 20 .mu.g/ml peptide in
phosphate buffered saline (PBS) overnight at 4.degree. C. The
plates are then washed and blocked with 200 .mu.l/well 2% bovine
serum albumin (BSA) in PBS. Sera are diluted in 2% BSA in PBS.
After overnight incubation at 4.degree. C. the plates are washed.
Bound murine IgG is detected by alkaline phosphatase
conjugated-goat anti-murine IgG (Jackson Immunoresearch
Laboratories) followed by p-nitrophenylphosphate substrate. The
titration curves for each sera are compared using DeltaSOFT II v.
3.66 (Biometallics, Princeton, N.J.).
[0247] Mice that develop sufficiently high titers of antibody that
bind to the peptide will be tested for specificity to frizzled 2 by
fluorescent cytometry with cells that express the protein by
transfection and known tumor cells that have the mRNA. We will also
test the binding by Western blot analysis of cells that express
this isoform and to cells that have been found to express other
frizzled family members. Briefly, immunoblotting will be performed
as described above. Cells are lysed in with a solution containing
25 mM Tris HCl, 150 mM KCl, 5 mM EDTA, 1% NP-40, 0.5% sodium
deoxycholic acid, 0.1% sodium dodecyl sulfate, 1 mM NaVO.sub.3, 1
mM NaF, 20 mM .beta.-glycerophosphate and protease inhibitors.
Twenty .mu.g of protein from each cell line is separated by
SDS-PAGE and transferred to a PVDF membrane. The membrane is soaked
in 2% I-block, 0.05% Tween X in PBS and then incubated with a 1:500
dilution of polyclonal pre or post immunization mouse serum at
1:500 dilution. Murine antibody binding is then detected by
horseradish peroxidase-conjugated rat anti-mouse IgG and
chemiluminescence (ECL detection reagents). To verify relative
amount of protein transferred in each lane, the blots are then
stripped and the presence of actin is measured with an actin
monoclonal antibody.
[0248] Different immunization strategies are being evaluated for
their efficacy in eliciting a humoral immune response. If the
antibody response is weak then the vectors can be redesigned with
other known potent T helper epitopes. Other vectors can be designed
where the polypeptide from frizzled 2 is shorter and does not
contain the cysteines, which may be inhibiting the most desirable
conformation. Another immunization strategy will be to use a prime
boost method. The animals are originally injected with plasmid DNA
and then are boosted with peptide or recombinant protein in
incomplete Freund's adjuvant. The B-cell epitope in each construct
may need to be redesigned until there is no cross-reactivity in the
humoral response to other frizzled isoforms.
Example 2
Expression of Wnt and Fzd mRNAs in HNSCC
[0249] Ten different HNSCC cell lines, two normal human
broncho-epithelial (NHBE) cell lines, and normal oral squamous
epithelial cells were tested by RT-PCR for the expression of five
Wnts (Wnt-1, Wnt-5a, Wnt-7a, Wnt-10b, Wnt-13), and two Fzds (Fzd-2
and 5). Representative results are illustrated in FIG. 8 and are
summarized in Table 1. When compared to the housekeeping gene
G3PDH, all the Wnts, as well as Fzd-2, were expressed more
frequently in HNSCC than in normal cells, while there was no
difference in Fzd-5 gene expression. Of the Wnt genes, Wnt-1, 5a,
and 10b were most strongly expressed by the malignant cells, but
were barely detectable in the normal tissues tested. We then
investigated further Wnt-1 and Wnt-10b, since these Wnts signal
through the canonical .beta.-catenin and LEF/TCF, and because
antibodies to the extracellular domains were available.
Example 3
Expression of Wnt/Fzd Proteins in HNSCC
[0250] Cell lines were lysed and analyzed for Wnt-1, Wnt-10b,
Fzd-2, and .beta.-catenin protein expression by immunoblotting
(FIG. 9). The normal cells expressed much less of these Wnt or Fzd
proteins, when compared to the tested HNSCC, with the exception of
RPMI 2650. Of note is the lack of detectable Fzd protein in the
lysate of the NHBE cell line that had a weakly detectable product
by RT-PCR. Beta-catenin was detected in all the samples, including
both HNSCC and NHBE lines.
Example 4
Effects of Anti-Wnt Antibodies and SFRP1
[0251] Treatment with antibody against the extracellular domains of
Wnt-1 or Wnt-10b decreased the proliferation of the SNU1076 HNSCC
cell line (FIG. 10), while little effect was observed in PCI 13
cells (data not shown). The inhibition of cell growth by the
antibodies was dependent on the concentration and incubation time.
The treatment of the SNU1076 HNSCC cell line with anti-Wnt
antibodies, but not control antibody, also induced apoptosis (FIG.
12). Similar to anti-Wnt antibodies, treatment with recombinant
SFRPI protein (2 .mu.g/ml), a natural antagonist of Wnt signaling,
inhibited growth of SNU 1076 cells (FIG. 11).
[0252] To determine if the effects of anti-Wnt antibody on SNU1076
cells were related to inhibition of Wnt signaling, we compared
levels of the Wnt regulated genes cyclin D1 and fibronectin (FIG.
7A). The anti-Wnt-1 antibody, but not the control IgG, reduced
cyclin D1, fibronectin, and .beta.-catenin levels in the cytosol of
SNU 1076 cells. To confirm these results, TOPFLASH-Luc, a reporter
plasmid containing TCF/LEF binding sites, or FOPFLASH-Luc, a
negative control plasmid having mutant binding sites was introduced
into SNU 1076 cells together with the pCMV-.beta.-gal plasmid (to
assess transfection efficiency). Luciferase activity was higher in
the TOPFLASH than the FOPFLASH transfected cells, indicating that
LEF/TCF dependent transcription was constitutively active. Cells
transfected with FOPFLASH showed no changes in the low baseline
luciferase activity after treatment with anti-Wnt1 antibodies,
whereas cells transfected with TOPFLASH displayed decreased
luciferase activity (FIG. 7B).
Example 5
Effects of Anti-Frizzled Antibodies
[0253] Wnt signaling through frizzled receptors has been described
to inhibit apoptosis (Chen, S. et al. "Wnt-1 signaling inhibits
apoptosis by activating beta-catenin/T cell factor-mediated
transcription,"J Cell Biol 152:87-96 (2001)). Also some of the
genes that are regulated by TCF/beta-catenin are known to be
associated with the cell cycle and cellular proliferation. By
blocking the binding of Wnt proteins to their receptors via
antibodies directed to the extracellular portion of frizzled this
pathway can be interrupted. Decreasing the downstream translocation
of beta-catenin to the nucleus could result in slower tumor growth
or death of the cell.
[0254] The immunization strategy that may be useful in terms of
raising specific antibodies that delay growth in cell culture will
then be tested for potential in vivo efficacy in mice. Previously
we have used the H-2.sup.b thymoma line EL4 as a syngeneic tumor in
C57B1/6 mice (Corr, M. et al., "Costimulation provided by DNA
immunization enhances antitumor immunity,"J Immunol 159:4999-5004
(1997); (Cho, H. J. et al., "Immunostimulatory DNA-based vaccines
induce cytotoxic lymphocyte activity by a T-helper cell-independent
mechanism," Nat Biotechnol 18:509-514 (2000)). This line will be
transfected with a human frizzled 2 expression vector and selected
in neomycin. The expression vector will be made by excising the
frizzled 2 containing insert from one expression vector with Ndel
and BamHl and ligating the insert into pcDNA3 (Invitrogen) which
has a CMV promoter and a neomycin selection cassette. Thirty-two
female C57B1/6 mice will be divided into groups of 8 mice each.
They will be injected in the dermis of the tail with a combination
of 50 .mu.g plasmid encoding a costimulator and 50 .mu.g linker
plasmid diluted in normal saline at weeks zero, one and two. A
group with empty vector is included as a negative control. On day
28 the mice will be injected subcutaneously in the flank with
20.times.10.sup.6 frizzled 2 transfected EL4 cells or untransfected
cells (Cho, H. J. et al., "Immunostimulatory DNA-based vaccines
induce cytotoxic lymphocyte activity by a T-helper cell-independent
mechanism," Nat Biotechnol 18:509-514 (2000)). The mice will be
monitored three times a week for weight, and tumor growth measured
with a caliper. Tumor volume is calculated by
length.times.width.sup.2.pi./6 as previously described (Radulovic,
S. et al., "Inhibition of growth of HT-29 human colon cancer
xenografls in nude mice by treatment with bombesin/gastrin
releasing peptide antagonist (RC-3095)," Cancer Res 51:6006-6009
(1991)). Mice will be sacrificed four weeks post tumor challenge or
if the tumor burden reaches approximately 2000 mm.sup.3. Inhibition
of tumor growth will be determined by ANOVA.
[0255] The polyclonal antibodies that are generated by the
immunization strategies may exhibit binding, but may not be
sufficiently concentrated in the polyclonal serum to have a
biologic effect. The serum from several immunization strategies may
need to be tested in vitro for their potential therapeutic utility
before proceeding with the in vivo active immunization strategy for
tumor prevention. The inhibition of tumor growth in the murine
model may be due to cellular responses as well as humoral, which
will lead to further investigations. These assays may be useful in
determining if the frizzled expressing cell lines are susceptible
to anti-proliferative activity of polyclonal anti-frizzled IgG.
Example 6
Overexpression of Wnt 14 and 16
[0256] Based upon sequences in the public human DNA gene database,
we prepared gene-specific primers for all the known human Wnt and
frizzled genes. We obtained mRNA from primary human chronic
lymphocytic leukemia cells or normal human lymphocytes. Using real
time PCR, we then compared the relative expression of the Wnt and
frizzled genes in the normal and malignant lymphocytes, compared to
the control genes GAPDH and 18S mRNA. We discovered that Wnt 16 was
70-100 fold overexpressed in the malignant lymphocytes. Wnt 14 was
400 fold overexpressed in the malignant lymphocytes. We sequenced
the amplicons to determine their identities. Northern blots of
normal human tissues confirmed the lack of significant expression
of Wnt 16 mRNA in non-lymphoid cells and in peripheral blood
lymphocytes. Following the procedures described above, we have
confirmed the overexpression of Wnt 16 in the malignant cells using
non-crossreactive antibodies and will confirm overexpression of Wnt
14 in a similar fashion. We have tested the effects of the anti-Wnt
16 antibodies on cell survival in vitro, using normal lymphocytes
as a control and will test anti-Wnt 14 antibodies in a similar
fashion. In addition, upon review of our results, we can develop
these antibodies and antigens as therapeutic agents.
Example 7
Regulation of Lymphocyte Survival by Integrins
[0257] The survival of lymphocytes requires that they interact with
the extracellular matrix proteins produced by stromal cells in
their surrounding micro environment. These interactions may render
the cells resistant to spontaneous and drug-induced apoptosis. VLA4
integrin-mediated cell adhesion is known to be involved in
regulating cell survival in some leukemic cell lines. We are
studying integrin effects on the survival of primary blood
lymphocytes. Our data show that the .alpha.4-CS1 fragment of
fibronectin significantly improves the survival of blood
lymphocytes. To develop a potential therapeutic strategy that
combines integrin antagonists with cytotoxic drugs, we are
investigating the mechanism of several integrin .alpha.4-specific
antagonists. These compounds specifically inhibit the adhesion of B
chronic lymphocytic leukemia cells to fibronectin. We are currently
studying the signaling events affected by these integrin
antagonists in primary human lymphocytes.
Example 8
Wnt Gene Expression in Normal and Malignant Lymphocytes
[0258] The secreted proteins of the diverse Wnt gene family are
known to play an important role in cell growth and differentiation.
Evidence suggests that Wnt signaling may regulate apoptosis.
Experiments described below were designed to identify the Wnt genes
that are most highly expressed in resting lymphocytes, and then to
determine their potential role in cell survival.
[0259] Total RNA was prepared and treated with RNase-free DNase.
The cDNA was synthesized from 5 .mu.g total RNA using Superscript
reverse transcriptase and oligo dT. To assure that there was no
genomic DNA contamination, controls in which no reverse
transcriptase was added were also carried out. TaqMan real-time PCR
was performed using an ABI PRISM 7700 sequence Detector. Primers
and probes for 46 Wnt family members and their related genes were
designed using Primer Express version 1.0 (Applied Biosystems). The
primers are shown in FIGS. 13A and 13B. The reaction conditions
were as follows: 2 min at 50.degree. C. (one cycle), 10 min at
95.degree. C. (one cycle), and 15s at 95.degree. C. and 1 min at
60.degree. C. (45 cycles). Two replicates for each gene were
performed.
[0260] Having developed and validated a TaqMan real-time PCR assay
to quantify the gene expression profiles of the wnt family and its
related genes, we measured the gene expression profile in three
B-CLL, two normal peripheral blood lymphocyte populations, and one
purified B cell sample. We found that wnt6, wnt14 and wnt16 were
overexpressed in B-CLL, compared to normal PBL or purified B cells.
Wnt14 mRNA levels in B-CLL were 16-178 times those of PBL and B
cell samples. The concentration of wnt6 mRNA in B-CLL samples was
8-32 fold higher than that in normal PBL and B-CLL samples. Wnt16
mRNA was expressed at 32-178 higher levels in B-CLL than in PBL.
For other Wnt-related families, such as Fzd, Frp, Wisp and DKK, we
did not observe any significant differences. Thus, the Wnt gene
overexpression appears to be unique.
[0261] We have established a model system to study the
integrin-dependent interaction of primary human lymphocytes with
extracellular matrix proteins, and have shown that the binding
promotes cell survival. We can now test the effects of integrin
antagonists on cell signaling and apoptosis in both normal and
malignant cells.
[0262] Other experiments revealed three wnt genes that are
overexpressed in lymphocytes of patients with B-CLL, compared to
normal peripheral blood lymphocytes. Since wnt proteins are
secreted, they may function as survival factors for the malignant
cells.
[0263] The specificities of the feeder cell-lymphocyte interactions
that delay senescence and apoptosis are identified by using
purified lymphocyte subpopulations (CD4, T cells, CD8, T cells, B
cells), co-culturing with different feeder cells (monocytes,
dendritic cells, endothelial cells, fibroblasts), and then
measuring both spontaneous and drug-induced apoptosis.
[0264] The specific surface molecules and/or secreted factors
responsible for the extended survival of the lymphocytes are
identified by testing the effects of blocking antibodies against
surface antigens on the feeder cells and the lymphocytes,
determining the effect of neutralizing antibodies against cytokines
and growth factors, and generating sense and anti-sense
transfectomas of feeder cells to confirm the roles of the specific
interaction revealed in the first two methods described.
[0265] The intracellular signaling pathways in quiescent
lymphocytes that are altered by contact with feeder cells, and that
increase their survival are identified by determining levels and
phosphorylation status of proteins in key activation pathways
(mitogen activated protein kinase, STATs, NF-Kb, b-catenin),
assessing levels and phosphorylation status of proteins that
regulate apoptosis (bcl2 family members, caspases, IAPs,
SMAC/DIABLO), and testing the effects of pharmacologic inhibitors
of signal transduction on the survival of quiescent lymphocytes
cultivated with feeder cells, alone or in combination with
cytotoxic agents.
Example 9
Expression of Wnt and Fzd Genes in Primary Breast Cancer Tumors and
CLL Cells
[0266] Wnt and Fzd levels were compared in primary cancer cells
from breast cancer tumors and CLL cells. Results are shown in FIGS.
14-27. Primers and hybridization probes are shown in FIGS. 13A and
13B. Gene expression levels were determined using real time PCR.
Briefly, total RNA was isolated from microdissected tissues using
RNA STAT-60, and reverse transcribed using random hexamer primers
and a Superscript Preamplification System. Then real time PCR was
performed using 18S RNA as a control gene. PCR was performed in a
Taqman Universal PCR MasterMix with initial activation at
95.degree. C. for 15 sec and 60.degree. C. for 10 min, and 40
cycles of 95.degree. C. for 15 sec and 60.degree. C. for 1 min. The
fluorescent signal was plotted versus cycle number, and the
threshold cycle was determined. Dilutions of cDNA from a pool of 12
normal tissues served as a positive control. The relative message
levels were calculated relative to standard calibration curves,
which were used in common by the different components of the SCOR,
and coordinated by the Research Resources Core. The pairwise
comparisons between different sites in the tested tissues were used
for statistical analyses. FIGS. 13A and 13B show the primer sets
and the Taqman hybridization probes for the analyzed genes. These
primers were previously validated in analyses of normal human
tissues and unfractionated RA synovial specimens
[0267] The data in the figures are relative, with the lowest normal
tissue level assigned a value of one. Thus, a relative value of 100
in breast cancer tumor or CLL cells means that the cancer cells had
100 times the values of the lowest normal tissue, as reported by
real time PCR.
[0268] CLL cells have high wnt16 levels, and also over-express
wnt3. (FIG. 14.) CLL cells also express Fzd 3. (FIGS. 16-17.)
[0269] Breast cancer cells expressed very high wnt7b levels. (FIGS.
14 and 27.) Other specific Wnts that are expressed at levels
greater than 5 times normal cell levels in breast tumors include
wnt 5a, wnt 10b, and wnt 14. (Id.) Breast cancer tumors also
expressed specific Fzd at high levels, including fzd 3, fzd 4, fzd
6, fzd 7, and fzd 10. (FIGS. 16-19)
[0270] Expression levels of Wnt downstream signaling genes and Wnt
antagonists were also determined in normal cells, CLL cells, and
primary breast tumors. The levels of these genes correlate with
activity of the expressed wnt genes and proteins. DKKs and FRP 2/4
are antagonists of wnt/fzd signaling pathway that bind either the
wnts or the frizzled co-receptor LRP5. DKK levels were increased in
some breast cancer tumors. (FIG. 20.) FRP 2/4 were overexpressed in
some breast cancer tumors. (FIG. 21.)
[0271] Wnt inducible genes include cyclin D1, c-myc, and the
WISP's. WISP refers to wnt-inducible serum protein and WISP2 refers
to wnt-inducible serum protein 2. WISP2 expression was increased in
breast tumor cells. (FIG. 27.) Cyclin D1 and c-myc levels were also
high relative to expression levels in normal cells. (FIGS. 23-24
and 27.) Cyclin D1 levels were also elevated in CLL cells relative
to normal lymphocytes. (FIGS. 23-24.) This indicates that the
specific wnt and fzd expression seen in breast tumors leads to
induction of genes and proteins downstream from the wnt/fzd signal.
That is, the specific wnt and fzd proteins are active and their
expression results in signal transduction. Levels of control gene
products IL-6 and MMP3, a breast cancer marker were also
determined. (FIGS. 25-27.)
[0272] Because some genes induced by a specific wnt/fzd signal are
required for proliferation in specific cell types, including cancer
cells, blocking a specific wnt/fzd signal by blocking binding of
the two molecules can be used to inhibit cellular proliferation.
For example, cyclin D1 is required for passage through the G1/S
transition in some cells types. Thus, antibodies against a specific
wnt or fzd, alone or in combination, can be used to block a
specific wnt/fzd interaction resulting in inhibition of cell
proliferation or in some case induction of apoptosis.
[0273] Levels of wnt 5a, wnt 7b, wnt 10b, and wnt 14 expression are
determined in samples from breast cancer tumors. If a wnt gene or
protein is highly expressed, antibodies against the overexpressed
gene product (e.g., wnt5a, wnt7b, wnt 10b, or wnt 14) are
administered to the patient. Administration of the wnt-specific
antibody blocks wnt signaling and can result in diminished or no
expression of required downstream wnt regulated genes and protein,
(e.g., cyclin D1, c-myc, and members of the WISP family), leading
to the death of the breast cancer cells. In some instances the
wnt-specific antibody is radiolabeled or conjugated to a toxin to
facilitate killing of the cancer cells. Induction of the complement
cascade or radiolabeled or toxin-conjugated antibodies are used to
kill breast cancer cells that overexpress a specific wnt, but do
not require on that specific wnt for proliferation or survival or
prliferation.
[0274] Levels of fzd 3, fzd 4, fzd 6, fzd 7, and fzd 10 expression
are determined in samples from breast cancer tumors. If a fzd gene
or protein is highly expressed, antibodies against the
overexpressed gene product (e.g., fzd 3, fzd 4, fzd 6, fzd 7, and
fzd 10) are administered to the patient. Administration of the
fzd-specific antibody blocks fzd signaling and can result in
diminished or no expression of required downstream fzd regulated
genes and protein, (e.g., cyclin D1, c-myc, and members of the WISP
family), leading to the death of the breast cancer cells. In some
instances the fzd-specific antibody is radiolabeled or conjugated
to a toxin to facilitate killing of the cancer cells. Induction of
the complement cascade or radiolabeled or toxin-conjugated
antibodies are used to kill breast cancer cells that overexpress a
specific fzd, but do not rely on that specific fzd for
survival.
[0275] Levels of wnt16 and wnt3 expression are determined in
samples from CLL cells. If a wnt16 or a wnt3 gene or protein is
highly expressed, antibodies against the overexpressed gene product
(e.g., wnt16 or wnt3) are administered to the patient.
Administration of the wnt-specific antibody blocks wnt signaling
and can result in diminished or no expression of required
downstream wnt regulated genes and protein, leading to the death of
the CLL cells. In some instances the wnt-specific antibody is
radiolabeled or conjugated to a toxin to facilitate killing of the
cancer cells. Induction of the complement cascade or radiolabeled
or toxin-conjugated antibodies are used to kill CLL cells that
overexpress a specific wnt, but do not rely on that specific wnt
for survival.
[0276] Levels of Fzd 3 expression are determined in samples from
CLL cells. If a Fzd 3 gene or protein is highly expressed,
antibodies against the overexpressed gene product (e.g., Fzd 3) are
administered to the patient. Administration of the Fzd 3-specific
antibody blocks Fzd 3 signaling can result in diminished or no
expression of required downstream fzd regulated genes and protein,
leading to the death of the CLL cells. In some instances the Fzd
3-specific antibody is radiolabeled or conjugated to a toxin to
facilitate killing of the cancer cells. Induction of the complement
cascade or radiolabeled or toxin-conjugated antibodies are used to
kill CLL cells that overexpress a specific fzd, but do not rely on
that specific fzd for survival.
Example 10
Expression of Wnt and Fzd Genes in Human Tonsils and Mantle Zone
Lymphomas
[0277] Normal human tonsil were stained using the anti-wnt16
antibodies. The antibodies stained mainly the B cells in the mantle
zone and the germinal centers, that are thought to be immature or
activated B cells. (Data not shown.)
[0278] Mantle zone lymphomas are an incurable, aggressive B cell
neoplasm, and represent a target for the specific wnt16 antibody.
Mantle zone lymphoma cells are assayed for wnt16 expression using
real time PCR as described above and using wnt16 specific
antibodies. Mantle zone lymphomas that overexpress wnt16 relative
to normal B cells or relative to expression of other specific wnts
in the mantle zone lymphoma cells. Mantle zone lymphomas that
overexpress a specific wnt16 are treated with wnt16 specific
antibodies to inhibit proliferation of lymphoma cells that rely on
expression of a downstream wnt/fzd induced gene. In some cases the
mantle zone lymphoma cells are treated with radiolabeled or
toxin-conjugated wnt16 specific antibodies to inhibit cellular
proliferation or induce apoptosis of the mantle zone lymphoma
cells.
[0279] Numerous modifications may be made to the foregoing systems
without departing from the basic teachings thereof. Although the
present invention has been described in substantial detail with
reference to one or more specific embodiments, those of skill in
the art will recognize that changes may be made to the embodiments
specifically disclosed in this application, yet these modifications
and improvements are within the scope and spirit of the invention,
as set forth in the claims which follow. All publications or patent
documents cited in this specification are incorporated herein by
reference as if each such publication or document was specifically
and individually indicated to be incorporated herein by
reference.
[0280] Citation of the above publications or documents is not
intended as an admission that any of the foregoing is pertinent
prior art, nor does it constitute any admission as to the contents
or date of these publications or documents.
Sequence CWU 1
1
232 1 370 PRT Homo sapiens human Wnt-1 1 Met Gly Leu Trp Ala Leu
Leu Pro Gly Trp Val Ser Ala Thr Leu Leu 1 5 10 15 Leu Ala Leu Ala
Ala Leu Pro Ala Ala Leu Ala Ala Asn Ser Ser Gly 20 25 30 Arg Trp
Trp Gly Ile Val Asn Val Ala Ser Ser Thr Asn Leu Leu Thr 35 40 45
Asp Ser Lys Ser Leu Gln Leu Val Leu Glu Pro Ser Leu Gln Leu Leu 50
55 60 Ser Arg Lys Gln Arg Arg Leu Ile Arg Gln Asn Pro Gly Ile Leu
His 65 70 75 80 Ser Val Ser Gly Gly Leu Gln Ser Ala Val Arg Glu Cys
Lys Trp Gln 85 90 95 Phe Arg Asn Arg Arg Trp Asn Cys Pro Thr Ala
Pro Gly Pro His Leu 100 105 110 Phe Gly Lys Ile Val Asn Arg Gly Cys
Arg Glu Thr Ala Phe Ile Phe 115 120 125 Ala Ile Thr Ser Ala Gly Val
Thr His Ser Val Ala Arg Ser Cys Ser 130 135 140 Glu Gly Ser Ile Glu
Ser Cys Thr Cys Asp Tyr Arg Arg Arg Gly Pro 145 150 155 160 Gly Gly
Pro Asp Trp His Trp Gly Gly Cys Ser Asp Asn Ile Asp Phe 165 170 175
Gly Arg Leu Phe Gly Arg Glu Phe Val Asp Ser Gly Glu Lys Gly Arg 180
185 190 Asp Leu Arg Phe Leu Met Asn Leu His Asn Asn Glu Ala Gly Arg
Thr 195 200 205 Thr Val Phe Ser Glu Met Arg Gln Glu Cys Lys Cys His
Gly Met Ser 210 215 220 Gly Ser Cys Thr Val Arg Thr Cys Trp Met Arg
Leu Pro Thr Leu Arg 225 230 235 240 Ala Val Gly Asp Val Leu Arg Asp
Arg Phe Asp Gly Ala Ser Arg Val 245 250 255 Leu Tyr Gly Asn Arg Gly
Ser Asn Arg Ala Ser Arg Ala Glu Leu Leu 260 265 270 Arg Leu Glu Pro
Glu Asp Pro Ala His Lys Pro Pro Ser Pro His Asp 275 280 285 Leu Val
Tyr Phe Glu Lys Ser Pro Asn Phe Cys Thr Tyr Ser Gly Arg 290 295 300
Leu Gly Thr Ala Gly Thr Ala Gly Arg Ala Cys Asn Ser Ser Ser Pro 305
310 315 320 Ala Leu Asp Gly Cys Glu Leu Leu Cys Cys Gly Arg Gly His
Arg Thr 325 330 335 Arg Thr Gln Arg Val Thr Glu Arg Cys Asn Cys Thr
Phe His Trp Cys 340 345 350 Cys His Val Ser Cys Arg Asn Cys Thr His
Thr Arg Val Leu His Glu 355 360 365 Cys Leu 370 2 2368 DNA Homo
sapiens human Wnt-1 2 gcggtgccgc ccgccgtggc cgcctcagcc caccagccgg
gaccgcgagc catgctgtcc 60 gccgcccgcc cccagggttg ttaaagccag
actgcgaact ctcgccactg ccgccaccgc 120 cgcgtcccgt cccaccgtcg
cgggcaacaa ccaaagtcgc cgcaactgca gcacagagcg 180 ggcaaagcca
ggcaggccat ggggctctgg gcgctgttgc ctggctgggt ttctgctacg 240
ctgctgctgg cgctggccgc tctgcccgca gccctggctg ccaacagcag tggccgatgg
300 tggggtattg tgaacgtagc ctcctccacg aacctgctta cagactccaa
gagtctgcaa 360 ctggtactcg agcccagtct gcagctgttg agccgcaaac
agcggcgtct gatacgccaa 420 aatccgggga tcctgcacag cgtgagtggg
gggctgcaga gtgccgtgcg cgagtgcaag 480 tggcagttcc ggaatcgccg
ctggaactgt cccactgctc cagggcccca cctcttcggc 540 aagatcgtca
accgaggctg tcgagaaacg gcgtttatct tcgctatcac ctccgccggg 600
gtcacccatt cggtggcgcg ctcctgctca gaaggttcca tcgaatcctg cacgtgtgac
660 taccggcggc gcggccccgg gggccccgac tggcactggg ggggctgcag
cgacaacatt 720 gacttcggcc gcctcttcgg ccgggagttc gtggactccg
gggagaaggg gcgggacctg 780 cgcttcctca tgaaccttca caacaacgag
gcaggccgta cgaccgtatt ctccgagatg 840 cgccaggagt gcaagtgcca
cgggatgtcc ggctcatgca cggtgcgcac gtgctggatg 900 cggctgccca
cgctgcgcgc cgtgggcgat gtgctgcgcg accgcttcga cggcgcctcg 960
cgcgtcctgt acggcaaccg cggcagcaac cgcgcttcgc gagcggagct gctgcgcctg
1020 gagccggaag acccggccca caaaccgccc tccccccacg acctcgtcta
cttcgagaaa 1080 tcgcccaact tctgcacgta cagcggacgc ctgggcacag
caggcacggc agggcgcgcc 1140 tgtaacagct cgtcgcccgc gctggacggc
tgcgagctgc tctgctgcgg caggggccac 1200 cgcacgcgca cgcagcgcgt
caccgagcgc tgcaactgca ccttccactg gtgctgccac 1260 gtcagctgcc
gcaactgcac gcacacgcgc gtactgcacg agtgtctgtg aggcgctgcg 1320
cggactcgcc cccaggaaac gctctcctcg agccctcccc caaacagact cgctagcact
1380 caagacccgg ttattcgccc acccgagtac ctccagtcac actccccgcg
gttcatacgc 1440 atcccatctc tcccacttcc tcctacctgg ggactcctca
aaccacttgc ctggggcggc 1500 atgaaccctc ttgccatcct gatggacctg
ccccggacct acctccctcc ctctccgcgg 1560 gagacccctt gttgcactgc
cccctgcttg gccaggaggt gagagaagga tgggtcccct 1620 ccgccatggg
gtcggctcct gatggtgtca ttctgcctgc tccatcgcgc cagcgacctc 1680
tctgcctctc ttcttcccct ttgtcctgcg ttttctccgg gtcctcctaa gtcccttcct
1740 attctcctgc catgggtgca gaccctgaac ccacacctgg gcatcagggc
ctttctcctc 1800 cccacctgta gctgaagcag gaggttacag ggcaaaaggg
cagctgtgat gatgtggaaa 1860 tgaggttggg ggaaccagca gaaatgcccc
cattctccca gtctctgtcg tggagccatt 1920 gaacagctgt gagccatgcc
tccctgggcc acctcctacc ccttcctgtc ctgcctcctc 1980 atcagtgtgt
aaataatttg cactgaaacg tggatacaga gccacgagtt tggatgttgt 2040
aaataaaact atttattgtg ctgggtccca gcctggtttg caaagaccac ctccaaccca
2100 acccaatccc tctccactct tctctccttt ctccctgcag ccttttctgg
tccctcttct 2160 ctcctcagtt tctcaaagat gcgtttgcct cctggaatca
gtatttcctt ccactgtagc 2220 tattagcggc tcctcgcccc caccagtgta
gcatcttcct ctgcagaata aaatctctat 2280 ttttatcgat gacttggtgg
cttttccttg aatccagaac acaaccttgt ttgtggtgtc 2340 ccctatcctc
cccttttacc actcccag 2368 3 360 PRT Homo sapiens human Wnt-2 3 Met
Asn Ala Pro Leu Gly Gly Ile Trp Leu Trp Leu Pro Leu Leu Leu 1 5 10
15 Thr Trp Leu Thr Pro Glu Val Asn Ser Ser Trp Trp Tyr Met Arg Ala
20 25 30 Thr Gly Gly Ser Ser Arg Val Met Cys Asp Asn Val Pro Gly
Leu Val 35 40 45 Ser Ser Gln Arg Gln Leu Cys His Arg His Pro Asp
Val Met Arg Ala 50 55 60 Ile Ser Gln Gly Val Ala Glu Trp Thr Ala
Glu Cys Gln His Gln Phe 65 70 75 80 Arg Gln His Arg Trp Asn Cys Asn
Thr Leu Asp Arg Asp His Ser Leu 85 90 95 Phe Gly Arg Val Leu Leu
Arg Ser Ser Arg Glu Ser Ala Phe Val Tyr 100 105 110 Ala Ile Ser Ser
Ala Gly Val Val Phe Ala Ile Thr Arg Ala Cys Ser 115 120 125 Gln Gly
Glu Val Lys Ser Cys Ser Cys Asp Pro Lys Lys Met Gly Ser 130 135 140
Ala Lys Asp Ser Lys Gly Ile Phe Asp Trp Gly Gly Cys Ser Asp Asn 145
150 155 160 Ile Asp Tyr Gly Ile Lys Phe Ala Arg Ala Phe Val Asp Ala
Lys Glu 165 170 175 Arg Lys Gly Lys Asp Ala Arg Ala Leu Met Asn Leu
His Asn Asn Arg 180 185 190 Ala Gly Arg Lys Ala Val Lys Arg Phe Leu
Lys Gln Glu Cys Lys Cys 195 200 205 His Gly Val Ser Gly Ser Cys Thr
Leu Arg Thr Cys Trp Leu Ala Met 210 215 220 Ala Asp Phe Arg Lys Thr
Gly Asp Tyr Leu Trp Arg Lys Tyr Asn Gly 225 230 235 240 Ala Ile Gln
Val Val Met Asn Gln Asp Gly Thr Gly Phe Thr Val Ala 245 250 255 Asn
Glu Arg Phe Lys Lys Pro Thr Lys Asn Asp Leu Val Tyr Phe Glu 260 265
270 Asn Ser Pro Asp Tyr Cys Ile Arg Asp Arg Glu Ala Gly Ser Leu Gly
275 280 285 Thr Ala Gly Arg Val Cys Asn Leu Thr Ser Arg Gly Met Asp
Ser Cys 290 295 300 Glu Val Met Cys Cys Gly Arg Gly Tyr Asp Thr Ser
His Val Thr Arg 305 310 315 320 Met Thr Lys Cys Gly Cys Lys Phe His
Trp Cys Cys Ala Val Arg Cys 325 330 335 Gln Asp Cys Leu Glu Ala Leu
Asp Val His Thr Cys Lys Ala Pro Lys 340 345 350 Asn Ala Asp Trp Thr
Thr Ala Thr 355 360 4 2301 DNA Homo sapiens human Wnt-2 4
agcagagcgg acgggcgcgc gggaggcgcg cagagctttc gggctgcagg cgctcgctgc
60 cgctggggaa ttgggctgtg ggcgaggcgg tccgggctgg cctttatcgc
tcgctgggcc 120 catcgtttga aactttatca gcgagtcgcc actcgtcgca
ggaccgagcg gggggcgggg 180 gcgcggcgag gcggcggccg tgacgaggcg
ctcccggagc tgagcgcttc tgctctgggc 240 acgcatggcg cccgcacacg
gagtctgacc tgatgcagac gcaagggggt taatatgaac 300 gcccctctcg
gtggaatctg gctctggctc cctctgctct tgacctggct cacccccgag 360
gtcaactctt catggtggta catgagagct acaggtggct cctccagggt gatgtgcgat
420 aatgtgccag gcctggtgag cagccagcgg cagctgtgtc accgacatcc
agatgtgatg 480 cgtgccatta gccagggcgt ggccgagtgg acagcagaat
gccagcacca gttccgccag 540 caccgctgga attgcaacac cctggacagg
gatcacagcc tttttggcag ggtcctactc 600 cgaagtagtc gggaatctgc
ctttgtttat gccatctcct cagctggagt tgtatttgcc 660 atcaccaggg
cctgtagcca aggagaagta aaatcctgtt cctgtgatcc aaagaagatg 720
ggaagcgcca aggacagcaa aggcattttt gattggggtg gctgcagtga taacattgac
780 tatgggatca aatttgcccg cgcatttgtg gatgcaaagg aaaggaaagg
aaaggatgcc 840 agagccctga tgaatcttca caacaacaga gctggcagga
aggctgtaaa gcggttcttg 900 aaacaagagt gcaagtgcca cggggtgagc
ggctcatgta ctctcaggac atgctggctg 960 gccatggccg acttcaggaa
aacgggcgat tatctctgga ggaagtacaa tggggccatc 1020 caggtggtca
tgaaccagga tggcacaggt ttcactgtgg ctaacgagag gtttaagaag 1080
ccaacgaaaa atgacctcgt gtattttgag aattctccag actactgtat cagggaccga
1140 gaggcaggct ccctgggtac agcaggccgt gtgtgcaacc tgacttcccg
gggcatggac 1200 agctgtgaag tcatgtgctg tgggagaggc tacgacacct
cccatgtcac ccggatgacc 1260 aagtgtgggt gtaagttcca ctggtgctgc
gccgtgcgct gtcaggactg cctggaagct 1320 ctggatgtgc acacatgcaa
ggcccccaag aacgctgact ggacaaccgc tacatgaccc 1380 cagcaggcgt
caccatccac cttcccttct acaaggactc cattggatct gcaagaacac 1440
tggacctttg ggttctttct ggggggatat ttcctaaggc atgtggcctt tatctcaacg
1500 gaagccccct cttcctccct gggggcccca ggatgggggg ccacacgctg
cacctaaagc 1560 ctaccctatt ctatccatct cctggtgttc tgcagtcatc
tcccctcctg gcgagttctc 1620 tttggaaata gcatgacagg ctgttcagcc
gggagggtgg tgggcccaga ccactgtctc 1680 cacccacctt gacgtttctt
ctttctagag cagttggcca agcagaaaaa aaagtgtctc 1740 aaaggagctt
tctcaatgtc ttcccacaaa tggtcccaat taagaaattc catacttctc 1800
tcagatggaa cagtaaagaa agcagaatca actgcccctg acttaacttt aacttttgaa
1860 aagaccaaga cttttgtctg tacaagtggt tttacagcta ccacccttag
ggtaattggt 1920 aattacctgg agaagaatgg ctttcaatac ccttttaagt
ttaaaatgtg tatttttcaa 1980 ggcatttatt gccatattaa aatctgatgt
aacaaggtgg ggacgtgtgt cctttggtac 2040 tatggtgtgt tgtatctttg
taagagcaaa agcctcagaa agggattgct ttgcattact 2100 gtccccttga
tataaaaaat ctttagggaa tgagagttcc ttctcactta gaatctgaag 2160
ggaattaaaa agaagatgaa tggtctggca atattctgta actattgggt gaatatggtg
2220 gaaaataatt tagtggatgg aatatcagaa gtatatctgt acagatcaag
aaaaaaagga 2280 agaataaaat tcctatatca t 2301 5 314 PRT Homo sapiens
human Wnt-2b 5 Met Leu Asp Gly Leu Gly Val Val Ala Ile Ser Ile Phe
Gly Ile Gln 1 5 10 15 Leu Lys Thr Glu Gly Ser Leu Arg Thr Ala Val
Pro Gly Ile Pro Thr 20 25 30 Gln Ser Ala Phe Asn Lys Cys Leu Gln
Arg Tyr Ile Gly Ala Leu Gly 35 40 45 Ala Arg Val Ile Cys Asp Asn
Ile Pro Gly Leu Val Ser Arg Gln Arg 50 55 60 Gln Leu Cys Gln Arg
Tyr Pro Asp Ile Met Arg Ser Val Gly Glu Gly 65 70 75 80 Ala Arg Glu
Trp Ile Arg Glu Cys Gln His Gln Phe Arg His His Arg 85 90 95 Trp
Asn Cys Thr Thr Leu Arg Gly Asp Phe Asp Trp Gly Gly Cys Ser 100 105
110 Asp Asn Ile His Tyr Gly Val Arg Phe Ala Lys Ala Phe Val Asp Ala
115 120 125 Lys Glu Lys Arg Leu Lys Asp Ala Arg Ala Leu Met Asn Leu
His Asn 130 135 140 Asn Arg Cys Gly Arg Thr Ala Val Arg Arg Phe Leu
Lys Leu Glu Cys 145 150 155 160 Lys Cys His Gly Val Ser Gly Ser Cys
Thr Leu Arg Thr Cys Trp Arg 165 170 175 Ala Leu Ser Asp Phe Arg Arg
Thr Gly Asp Tyr Leu Arg Arg Arg Tyr 180 185 190 Asp Gly Ala Val Gln
Val Met Ala Thr Gln Asp Gly Ala Asn Phe Thr 195 200 205 Ala Ala Arg
Gln Gly Tyr Arg Arg Ala Thr Arg Thr Asp Leu Val Tyr 210 215 220 Phe
Asp Asn Ser Pro Asp Tyr Cys Val Leu Asp Lys Ala Ala Gly Ser 225 230
235 240 Leu Gly Thr Ala Gly Arg Val Cys Ser Lys Thr Ser Lys Gly Thr
Asp 245 250 255 Gly Cys Glu Ile Met Cys Cys Gly Arg Gly Tyr Asp Thr
Thr Arg Val 260 265 270 Thr Arg Val Thr Gln Cys Glu Cys Lys Phe His
Trp Cys Cys Ala Val 275 280 285 Arg Cys Lys Glu Cys Arg Asn Thr Val
Asp Val His Thr Cys Lys Ala 290 295 300 Pro Lys Lys Ala Glu Trp Leu
Asp Gln Thr 305 310 6 2014 DNA Homo sapiens human Wnt-2b 6
aaaccctgaa gagcccaagc aatgtggttg taaaatttgc aaaataagat taaatcttaa
60 ctgcaatctg ttaacactgc tgtctccttt cactctttct cctatatcac
actttcccac 120 atgttggatg gccttggagt ggtagccata agcatttttg
gaattcaact aaaaactgaa 180 ggatccttga ggacggcagt acctggcata
cctacacagt cagcgttcaa caagtgtttg 240 caaaggtaca ttggggcact
gggggcacga gtgatctgtg acaatatccc tggtttggtg 300 agccggcagc
ggcagctgtg ccagcgttac ccagacatca tgcgttcagt gggcgagggt 360
gcccgagaat ggatccgaga gtgtcagcac caattccgcc accaccgctg gaactgtacc
420 accctggacc gggaccacac cgtctttggc cgtgtcatgc tcagaagtag
ccgagaggca 480 gcttttgtat atgccatctc atcagcaggg gtagtccacg
ctattactcg cgcctgtagc 540 cagggtgaac tgagtgtgtg cagctgtgac
ccctacaccc gtggccgaca ccatgaccag 600 cgtggggact ttgactgggg
tggctgcagt gacaacatcc actacggtgt ccgttttgcc 660 aaggccttcg
tggatgccaa ggagaagagg cttaaggatg cccgggccct catgaactta 720
cataataacc gctgtggtcg cacggctgtg cggcggtttc tgaagctgga gtgtaagtgc
780 catggcgtga gtggttcctg tactctgcgc acctgctggc gtgcactctc
agatttccgc 840 cgcacaggtg attacctgcg gcgacgctat gatggggctg
tgcaggtgat ggccacccaa 900 gatggtgcca acttcaccgc agcccgccaa
ggctatcgcc gtgccacccg gactgatctt 960 gtctactttg acaactctcc
agattactgt gtcttggaca aggctgcagg ttccctaggc 1020 actgcaggcc
gtgtctgcag caagacatca aaaggaacag acggttgtga aatcatgtgc 1080
tgtggccgag ggtacgacac aactcgagtc acccgtgtta cccagtgtga gtgcaaattc
1140 cactggtgct gtgctgtacg gtgcaaggaa tgcagaaata ctgtggacgt
ccatacttgc 1200 aaagccccca agaaggcaga gtggctggac cagacctgaa
cacacagata cctcactcat 1260 ccctccaatt caagcctctc aactcaaaag
cacaagatcc ttgcatgcac accttcctcc 1320 accctccacc ctgggctgct
accgcttcta tttaaggatg tagagagtaa tccataggga 1380 ccatggtgtc
ctggctggtt ccttagccct gggaaggagt tgtcagggga tataagaaac 1440
tgtgcaagct ccctgatttc ccgctctgga gatttgaagg gagagtagaa gagatagggg
1500 gtctttagag tgaaatgagt tgcactaaag tacgtagttg aggctccttt
tttctttcct 1560 ttgcaccagc ttcccgacac ttcttggtgt gcaagaggaa
gggtacctgt agagagcttc 1620 tttttgtttc tacctggcca aagttagatg
ggacaaagat gaatggcatg tcccttctct 1680 gaagtccgtt tgagcagaac
tacctggtac cccgaaagaa aaatcttagg ctaccacatt 1740 ctattattga
gagcctgaga tgttagccat agtggacaag gttccattca catgctcata 1800
tgtttataaa ctgtgttttg tagaagaaaa agaatcataa caatacaaac acacattcat
1860 tctctctttt tctctctacc attctcaacc tgtattggac agcactgcct
cttttgctta 1920 cttgctgcct gttcaaactg aggtggaatg cagtggttcc
catgcttaac agatcattaa 1980 aacaccctag aacactccta ggatagatta atgt
2014 7 355 PRT Homo sapiens human Wnt-3 7 Met Glu Pro His Leu Leu
Gly Leu Leu Leu Gly Leu Leu Leu Gly Gly 1 5 10 15 Thr Arg Val Leu
Ala Gly Tyr Pro Ile Trp Trp Ser Leu Ala Leu Gly 20 25 30 Gln Gln
Tyr Thr Ser Leu Gly Ser Gln Pro Leu Leu Cys Gly Ser Ile 35 40 45
Pro Gly Leu Val Pro Lys Gln Leu Arg Phe Cys Arg Asn Tyr Ile Glu 50
55 60 Ile Met Pro Ser Val Ala Glu Gly Val Lys Leu Gly Ile Gln Glu
Cys 65 70 75 80 Gln His Gln Phe Arg Gly Arg Arg Trp Asn Cys Thr Thr
Ile Asp Asp 85 90 95 Ser Leu Ala Ile Phe Gly Pro Val Leu Asp Lys
Ala Thr Arg Glu Ser 100 105 110 Ala Phe Val His Ala Ile Ala Ser Ala
Gly Val Ala Phe Ala Val Thr 115 120 125 Arg Ser Cys Ala Glu Gly Thr
Ser Thr Ile Cys Gly Cys Asp Ser His 130 135 140 His Lys Gly Pro Pro
Gly Glu Gly Trp Lys Trp Gly Gly Cys Ser Glu 145 150 155 160 Asp Ala
Asp Phe Gly Val Leu Val Ser Arg Glu Phe Ala Asp Ala Arg 165 170 175
Glu Asn Arg Pro Asp Ala Arg Ser Ala Met Asn Lys His Asn Asn Glu 180
185 190 Ala Gly Arg Thr Thr Ile Leu Asp His Met His Leu Lys Cys Lys
Cys 195 200 205 His Gly Leu Ser Gly Ser Cys Glu Val Lys Thr Cys Trp
Trp Ala Gln 210 215 220 Pro Asp Phe Arg Ala Ile Gly Asp Phe Leu Lys
Asp Lys Tyr Asp Ser 225 230 235 240 Ala Ser Glu Met Val Val Glu Lys
His Arg Glu Ser Arg Gly Trp Val 245 250 255 Glu Thr Leu Arg Ala Lys
Tyr Ser Leu Phe Lys Pro Pro Thr Glu Arg
260 265 270 Asp Leu Val Tyr Tyr Glu Asn Ser Pro Asn Phe Cys Glu Pro
Asn Pro 275 280 285 Glu Thr Gly Ser Phe Gly Thr Arg Asp Arg Thr Cys
Asn Val Thr Ser 290 295 300 His Gly Ile Asp Gly Cys Asp Leu Leu Cys
Cys Gly Arg Gly His Asn 305 310 315 320 Thr Arg Thr Glu Lys Arg Lys
Glu Lys Cys His Cys Ile Phe His Trp 325 330 335 Cys Cys Tyr Val Ser
Cys Gln Glu Cys Ile Arg Ile Tyr Asp Val His 340 345 350 Thr Cys Lys
355 8 1506 DNA Homo sapiens human Wnt-3 8 gcgcttctga caagcccgaa
agtcatttcc aatctcaagt ggactttgtt ccaactattg 60 ggggcgtcgc
tccccctctt catggtcgcg ggcaaacttc ctcctcggcg cctcttctaa 120
tggagcccca cctgctcggg ctgctcctcg gcctcctgct cggtggcacc agggtcctcg
180 ctggctaccc aatttggtgg tccctggccc tgggccagca gtacacatct
ctgggctcac 240 agcccctgct ctgcggctcc atcccaggcc tggtccccaa
gcaactgcgc ttctgccgca 300 attacatcga gatcatgccc agcgtggccg
agggcgtgaa gctgggcatc caggagtgcc 360 agcaccagtt ccggggccgc
cgctggaact gcaccaccat agatgacagc ctggccatct 420 ttgggcccgt
cctcgacaaa gccacccgcg agtcggcctt cgttcacgcc atcgcctcgg 480
ccggcgtggc cttcgccgtc acccgctcct gcgccgaggg cacctccacc atttgcggct
540 gtgactcgca tcataagggg ccgcctggcg aaggctggaa gtggggcggc
tgcagcgagg 600 acgctgactt cggcgtgtta gtgtccaggg agttcgcgga
tgcgcgcgag aacaggccgg 660 acgcgcgctc ggccatgaac aagcacaaca
acgaggcggg ccgcacgact atcctggacc 720 acatgcacct caaatgcaag
tgccacgggc tgtcgggcag ctgtgaggtg aagacctgct 780 ggtgggcgca
gcctgacttc cgtgccatcg gtgacttcct caaggacaag tatgacagcg 840
cctcggagat ggtagtagag aagcaccgtg agtcccgagg ctgggtggag accctccggg
900 ccaagtactc gctcttcaag ccacccacgg agagggacct ggtctactac
gagaactccc 960 ccaacttttg tgagcccaac ccagagacgg gttcctttgg
cacaagggac cggacttgca 1020 atgtcacctc ccacggcatc gatggctgcg
atctgctctg ctgtggccgg ggccacaaca 1080 cgaggacgga gaagcggaag
gaaaaatgcc actgcatctt ccactggtgc tgctacgtca 1140 gctgccagga
gtgtattcgc atctacgacg tgcacacctg caagtagggc accagggcgc 1200
tgggaagggg tgaagtgtgt ggctgggcgg attcagcgaa gtctcatggg aagcaggacc
1260 tagagccggg cacagccctc agcgtcagac agcaaggaac tgtcaccagc
cgcacgcgtg 1320 gtaaatgacc cagacccaac tcgcctgtgg acggggaggc
tctccctctc tctcatctta 1380 catttctcac cctactctgg atggtgtgtg
gtttttaaag aagggggctt tctttttagt 1440 tctctagggt ctgataggaa
cagacctgag gcttatcttt gcacatgtta aagaaaaaaa 1500 aaaaaa 1506 9 352
PRT Homo sapiens human Wnt-3a 9 Met Ala Pro Leu Gly Tyr Phe Leu Leu
Leu Cys Ser Leu Lys Gln Ala 1 5 10 15 Leu Gly Ser Tyr Pro Ile Trp
Trp Ser Leu Ala Val Gly Pro Gln Tyr 20 25 30 Ser Ser Leu Gly Ser
Gln Pro Ile Leu Cys Ala Ser Ile Pro Gly Leu 35 40 45 Val Pro Lys
Gln Leu Arg Phe Cys Arg Asn Tyr Val Glu Ile Met Pro 50 55 60 Ser
Val Ala Glu Gly Ile Lys Ile Gly Ile Gln Glu Cys Gln His Gln 65 70
75 80 Phe Arg Gly Arg Arg Trp Asn Cys Thr Thr Val His Asp Ser Leu
Ala 85 90 95 Ile Phe Gly Pro Val Leu Asp Lys Ala Thr Arg Glu Ser
Ala Phe Val 100 105 110 His Ala Ile Ala Ser Ala Gly Val Ala Phe Ala
Val Thr Arg Ser Cys 115 120 125 Ala Glu Gly Thr Ala Ala Ile Cys Gly
Cys Ser Ser Arg His Gln Gly 130 135 140 Ser Pro Gly Lys Gly Trp Lys
Trp Gly Gly Cys Ser Glu Asp Ile Glu 145 150 155 160 Phe Gly Gly Met
Val Ser Arg Glu Phe Ala Asp Ala Arg Glu Asn Arg 165 170 175 Pro Asp
Ala Arg Ser Ala Met Asn Arg His Asn Asn Glu Ala Gly Arg 180 185 190
Gln Ala Ile Ala Ser His Met His Leu Lys Cys Lys Cys His Gly Leu 195
200 205 Ser Gly Ser Cys Glu Val Lys Thr Cys Trp Trp Ser Gln Pro Asp
Phe 210 215 220 Arg Ala Ile Gly Asp Phe Leu Lys Asp Lys Tyr Asp Ser
Ala Ser Glu 225 230 235 240 Met Val Val Glu Lys His Arg Glu Ser Arg
Gly Trp Val Glu Thr Leu 245 250 255 Arg Pro Arg Tyr Thr Tyr Phe Lys
Val Pro Thr Glu Arg Asp Leu Val 260 265 270 Tyr Tyr Glu Ala Ser Pro
Asn Phe Cys Glu Pro Asn Pro Glu Thr Gly 275 280 285 Ser Phe Gly Thr
Arg Asp Arg Thr Cys Asn Val Ser Ser His Gly Ile 290 295 300 Asp Gly
Cys Asp Leu Leu Cys Cys Gly Arg Gly His Asn Ala Arg Ala 305 310 315
320 Glu Arg Arg Arg Glu Lys Cys Arg Cys Val Phe His Trp Cys Cys Tyr
325 330 335 Val Ser Cys Gln Glu Cys Thr Arg Val Tyr Asp Val His Thr
Cys Lys 340 345 350 10 2932 DNA Homo sapiens human Wnt-3a 10
agctcccagg gcccggcccc ccccggcgct cacgctctcg gggcggactc ccggccctcc
60 gcgccctctc gcgcggcgat ggccccactc ggatacttct tactcctctg
cagcctgaag 120 caggctctgg gcagctaccc gatctggtgg tcgctggctg
ttgggccaca gtattcctcc 180 ctgggctcgc agcccatcct gtgtgccagc
atcccgggcc tggtccccaa gcagctccgc 240 ttctgcagga actacgtgga
gatcatgccc agcgtggccg agggcatcaa gattggcatc 300 caggagtgcc
agcaccagtt ccgcggccgc cggtggaact gcaccaccgt ccacgacagc 360
ctggccatct tcgggcccgt gctggacaaa gctaccaggg agtcggcctt tgtccacgcc
420 attgcctcag ccggtgtggc ctttgcagtg acacgctcat gtgcagaagg
cacggccgcc 480 atctgtggct gcagcagccg ccaccagggc tcaccaggca
agggctggaa gtggggtggc 540 tgtagcgagg acatcgagtt tggtgggatg
gtgtctcggg agttcgccga cgcccgggag 600 aaccggccag atgcccgctc
agccatgaac cgccacaaca acgaggctgg gcgccaggcc 660 atcgccagcc
acatgcacct caagtgcaag tgccacgggc tgtcgggcag ctgcgaggtg 720
aagacatgct ggtggtcgca acccgacttc cgcgccatcg gtgacttcct caaggacaag
780 tacgacagcg cctcggagat ggtggtggag aagcaccggg agtcccgcgg
ctgggtggag 840 accctgcggc cgcgctacac ctacttcaag gtgcccacgg
agcgcgacct ggtctactac 900 gaggcctcgc ccaacttctg cgagcccaac
cctgagacgg gctccttcgg cacgcgcgac 960 cgcacctgca acgtcagctc
gcacggcatc gacggctgcg acctgctgtg ctgcggccgc 1020 ggccacaacg
cgcgagcgga gcggcgccgg gagaagtgcc gctgcgtgtt ccactggtgc 1080
tgctacgtca gctgccagga gtgcacgcgc gtctacgacg tgcacacctg caagtaggca
1140 ccggccgcgg ctccccctgg acggggcggg ccctgcctga gggtgggctt
ttccctgggt 1200 ggagcaggac tcccacctaa acggggcagt actcctccct
gggggcggga ctcctccctg 1260 ggggtggggc tcctacctgg gggcagaact
cctacctgaa ggcagggctc ctccctggag 1320 ctagtgtctc ctctctggtg
gctgggctgc tcctgaatga ggcggagctc caggatgggg 1380 aggggctctg
cgttggcttc tccctgggga cggggctccc ctggacagag gcggggctac 1440
agattgggcg gggcttctct tgggtgggac agggcttctc ctgcgggggc gaggcccctc
1500 ccagtaaggg cgtggctctg ggtgggcggg gcactaggta ggcttctacc
tgcaggcggg 1560 gctcctcctg aaggaggcgg ggctctagga tggggcacgg
ctctggggta ggctgctccc 1620 tgagggcgga gcgcctcctt aggagtgggg
ttttatggtg gatgaggctt cttcctggat 1680 ggggcagagc ttctcctgac
cagggcaagg ccccttccac gggggctgtg gctctgggtg 1740 ggcgtggcct
gcataggctc cttcctgtgg gtggggcttc tctgggacca ggctccaatg 1800
gggcggggct tctctccgcg ggtgggactc ttccctggga accgccctcc tgattaaggc
1860 gtggcttctg caggaatccc ggctccagag caggaaattc agcccaccag
ccacctcatc 1920 cccaaccccc tgtaaggttc catccacccc tgcgtcgagc
tgggaaggtt ccatgaagcg 1980 agtcgggtcc ccaacccgtg cccctgggat
ccgagggccc ctctccaagc gcctggcttt 2040 ggaatgctcc aggcgcgccg
acgcctgtgc caccccttcc tcagcctggg gtttgaccac 2100 ccacctgacc
aggggcccta cctggggaaa gcctgaaggg cctcccagcc cccaacccca 2160
agaccaagct tagtcctggg agaggacagg gacttcgcag aggcaagcga ccgaggccct
2220 cccaaagagg cccgccctgc ccgggctccc acaccgtcag gtactcctgc
cagggaactg 2280 gcctgctgcg ccccaggccc cgcccgtctc tgctctgctc
agctgcgccc ccttctttgc 2340 agctgcccag cccctcctcc ctgccctcgg
gtctccccac ctgcactcca tccagctaca 2400 ggagagatag aagcctctcg
tcccgtccct ccctttcctc cgcctgtcca cagcccctta 2460 agggaaaggt
aggaagagag gtccagcccc ccaggctgcc cagagctgct ggtctcattt 2520
gggggcgttc gggaggtttg gggggcatca accccccgac tgtgctgctc gcgaaggtcc
2580 cacagccctg agatgggccg gcccccttcc tggcccctca tggcgggact
ggagaaatgg 2640 tccgctttcc tggagccaat ggcccggccc ctcctgactc
atccgcctgg cccgggaatg 2700 aatggggagg ccgctgaacc cacccggccc
atatccctgg ttgcctcatg gccagcgccc 2760 ctcagcctct gccactgtga
accggctccc accctcaagg tgcggggaga agaagcggcc 2820 aggcggggcg
ccccaagagc ccaaaagagg gcacaccgcc atcctctgcc tcaaattctg 2880
cgtttttggt tttaatgtta tatctgatgc tgctatatcc actgtccaac gg 2932 11
351 PRT Homo sapiens human Wnt-4 11 Met Ser Pro Arg Ser Cys Leu Arg
Ser Leu Arg Leu Leu Val Phe Ala 1 5 10 15 Val Phe Ser Ala Ala Ala
Ser Asn Trp Leu Tyr Leu Ala Lys Leu Ser 20 25 30 Ser Val Gly Ser
Ile Ser Glu Glu Glu Thr Cys Glu Lys Leu Lys Gly 35 40 45 Leu Ile
Gln Arg Gln Val Gln Met Cys Lys Arg Asn Leu Glu Val Met 50 55 60
Asp Ser Val Arg Arg Gly Ala Gln Leu Ala Ile Glu Glu Cys Gln Tyr 65
70 75 80 Gln Phe Arg Asn Arg Arg Trp Asn Cys Ser Thr Leu Asp Ser
Leu Pro 85 90 95 Val Phe Gly Lys Val Val Thr Gln Gly Thr Arg Glu
Ala Ala Phe Val 100 105 110 Tyr Ala Ile Ser Ser Ala Gly Val Ala Phe
Ala Val Thr Arg Ala Cys 115 120 125 Ser Ser Gly Glu Leu Glu Lys Cys
Gly Cys Asp Arg Thr Val His Gly 130 135 140 Val Ser Pro Gln Gly Phe
Gln Trp Ser Gly Cys Ser Asp Asn Ile Ala 145 150 155 160 Tyr Gly Val
Ala Phe Ser Gln Ser Phe Val Asp Val Arg Glu Arg Ser 165 170 175 Lys
Gly Ala Ser Ser Ser Arg Ala Leu Met Asn Leu His Asn Asn Glu 180 185
190 Ala Gly Arg Lys Ala Ile Leu Thr His Met Arg Val Glu Cys Lys Cys
195 200 205 His Gly Val Ser Gly Ser Cys Glu Val Lys Thr Cys Trp Arg
Ala Val 210 215 220 Pro Pro Phe Arg Gln Val Gly His Ala Leu Lys Glu
Lys Phe Asp Gly 225 230 235 240 Ala Thr Glu Val Glu Pro Arg Arg Val
Gly Ser Ser Arg Ala Leu Val 245 250 255 Pro Arg Asn Ala Gln Phe Lys
Pro His Thr Asp Glu Asp Leu Val Tyr 260 265 270 Leu Glu Pro Ser Pro
Asp Phe Cys Glu Gln Asp Met Arg Ser Gly Val 275 280 285 Leu Gly Thr
Arg Gly Arg Thr Cys Asn Lys Thr Ser Lys Ala Ile Asp 290 295 300 Gly
Cys Glu Leu Leu Cys Cys Gly Arg Gly Phe His Thr Ala Gln Val 305 310
315 320 Glu Leu Ala Glu Arg Cys Ser Cys Lys Phe His Trp Cys Cys Phe
Val 325 330 335 Lys Cys Arg Gln Cys Gln Arg Leu Val Glu Leu His Thr
Cys Arg 340 345 350 12 1198 DNA Homo sapiens human Wnt-4 12
gcggccgcag ccgctgcccc gggccgggcg cccgcggcgg caccatgagt ccccgctcgt
60 gcctgcgttc gctgcgcctc ctcgtcttcg ccgtcttctc agccgccgcg
agcaactggc 120 tgtacctggc caagctgtcg tcggtgggga gcatctcaga
ggaggagacg tgcgagaaac 180 tcaagggcct gatccagagg caggtgcaga
tgtgcaagcg gaacctggaa gtcatggact 240 cggtgcgccg cggtgcccag
ctggccattg aggagtgcca gtaccagttc cggaaccggc 300 gctggaactg
ctccacactc gactccttgc ccgtcttcgg caaggtggtg acgcaaggga 360
ctcgggaggc ggccttcgtg tacgccatct cttcggcagg tgtggccttt gcagtgacgc
420 gggcgtgcag cagtggggag ctggagaagt gcggctgtga caggacagtg
catggggtca 480 gcccacaggg cttccagtgg tcaggatgct ctgacaacat
cgcctacggt gtggccttct 540 cacagtcgtt tgtggatgtg cgggagagaa
gcaagggggc ctcgtccagc agagccctca 600 tgaacctcca caacaatgag
gccggcagga aggccatcct gacacacatg cgggtggaat 660 gcaagtgcca
cggggtgtca ggctcctgtg aggtaaagac gtgctggcga gccgtgccgc 720
ccttccgcca ggtgggtcac gcactgaagg agaagtttga tggtgccact gaggtggagc
780 cacgccgcgt gggctcctcc agggcactgg tgccacgcaa cgcacagttc
aagccgcaca 840 cagatgagga cctggtgtac ttggagccta gccccgactt
ctgtgagcag gacatgcgca 900 gcggcgtgct gggcacgagg ggccgcacat
gcaacaagac gtccaaggcc atcgacggct 960 gtgagctgct gtgctgtggc
cgcggcttcc acacggcgca ggtggagctg gctgaacgct 1020 gcagctgcaa
attccactgg tgctgcttcg tcaagtgccg gcagtgccag cggctcgtgg 1080
agttgcacac gtgccgatga ccgcctgcct agccctgcgc cggcaaccac ctagtggccc
1140 agggaaggcc gataatttaa acagtctccc accacctacc ccaagagata
ctggttgt 1198 13 365 PRT Homo sapiens human Wnt-5a 13 Met Ala Gly
Ser Ala Met Ser Ser Lys Phe Phe Leu Val Ala Leu Ala 1 5 10 15 Ile
Phe Phe Ser Phe Ala Gln Val Val Ile Glu Ala Asn Ser Trp Trp 20 25
30 Ser Leu Gly Met Asn Asn Pro Val Gln Met Ser Glu Val Tyr Ile Ile
35 40 45 Gly Ala Gln Pro Leu Cys Ser Gln Leu Ala Gly Leu Ser Gln
Gly Gln 50 55 60 Lys Lys Leu Cys His Leu Tyr Gln Asp His Met Gln
Tyr Ile Gly Glu 65 70 75 80 Gly Ala Lys Thr Gly Ile Lys Glu Cys Gln
Tyr Gln Phe Arg His Arg 85 90 95 Arg Trp Asn Cys Ser Thr Val Asp
Asn Thr Ser Val Phe Gly Arg Val 100 105 110 Met Gln Ile Gly Ser Arg
Glu Thr Ala Phe Thr Tyr Ala Val Ser Ala 115 120 125 Ala Gly Val Val
Asn Ala Met Ser Arg Ala Cys Arg Glu Gly Glu Leu 130 135 140 Ser Thr
Cys Gly Cys Ser Arg Ala Ala Arg Pro Lys Asp Leu Pro Arg 145 150 155
160 Asp Trp Leu Trp Gly Gly Cys Gly Asp Asn Ile Asp Tyr Gly Tyr Arg
165 170 175 Phe Ala Lys Glu Phe Val Asp Ala Arg Glu Arg Glu Arg Ile
His Ala 180 185 190 Lys Gly Ser Tyr Glu Ser Ala Arg Ile Leu Met Asn
Leu His Asn Asn 195 200 205 Glu Ala Gly Arg Arg Thr Val Tyr Asn Leu
Ala Asp Val Ala Cys Lys 210 215 220 Cys His Gly Val Ser Gly Ser Cys
Ser Leu Lys Thr Cys Trp Leu Gln 225 230 235 240 Leu Ala Asp Phe Arg
Lys Val Gly Asp Ala Leu Lys Glu Lys Tyr Asp 245 250 255 Ser Ala Ala
Ala Met Arg Leu Asn Ser Arg Gly Lys Leu Val Gln Val 260 265 270 Asn
Ser Arg Phe Asn Ser Pro Thr Thr Gln Asp Leu Val Tyr Ile Asp 275 280
285 Pro Ser Pro Asp Tyr Cys Val Arg Asn Glu Ser Thr Gly Ser Leu Gly
290 295 300 Thr Gln Gly Arg Leu Cys Asn Lys Thr Ser Glu Gly Met Asp
Gly Cys 305 310 315 320 Glu Leu Met Cys Cys Gly Arg Gly Tyr Asp Gln
Phe Lys Thr Val Gln 325 330 335 Thr Glu Arg Cys His Cys Lys Phe His
Trp Cys Cys Tyr Val Lys Cys 340 345 350 Lys Lys Cys Thr Glu Ile Val
Asp Gln Phe Val Cys Lys 355 360 365 14 4428 DNA Homo sapiens human
Wnt-5a 14 ttaaggaaat ccgggctgct cttccccatc tggaagtggc tttccccaca
tcggctcgta 60 aactgattat gaaacatacg atgttaattc ggagctgcat
ttcccagctg ggcactctcg 120 cgcgctggtc cccggggcct cgccccccac
cccctgccct tccctcccgc gtcctgcccc 180 catcctccac cccccgcgct
ggccaccccg cctccttggc agcctctggc ggcagcgcgc 240 tccactcgcc
tcccgtgctc ctctcgccca tggaattaat tctggctcca cttgttgctc 300
ggcccaggtt ggggagagga cggagggtgg ccgcagcggg ttcctgagtg aattacccag
360 gagggactga gcacagcacc aactagagag gggtcagggg gtgcgggact
cgagcgagca 420 ggaaggaggc agcgcctggc accagggctt tgactcaaca
gaattgagac acgtttgtaa 480 tcgctggcgt gccccgcgca caggatccca
gcgaaaatca gatttcctgg tgaggttgcg 540 tgggtggatt aatttggaaa
aagaaactgc ctatatcttg ccatcaaaaa actcacggag 600 gagaagcgca
gtcaatcaac agtaaactta agagaccccc gatgctcccc tggtttaact 660
tgtatgcttg aaaattatct gagagggaat aaacatcttt tccttcttcc ctctccagaa
720 gtccattgga atattaagcc caggagttgc tttggggatg gctggaagtg
caatgtcttc 780 caagttcttc ctagtggctt tggccatatt tttctccttc
gcccaggttg taattgaagc 840 caattcttgg tggtcgctag gtatgaataa
ccctgttcag atgtcagaag tatatattat 900 aggagcacag cctctctgca
gccaactggc aggactttct caaggacaga agaaactgtg 960 ccacttgtat
caggaccaca tgcagtacat cggagaaggc gcgaagacag gcatcaaaga 1020
atgccagtat caattccgac atcgacggtg gaactgcagc actgtggata acacctctgt
1080 ttttggcagg gtgatgcaga taggcagccg cgagacggcc ttcacatacg
ccgtgagcgc 1140 agcaggggtg gtgaacgcca tgagccgggc gtgccgcgag
ggcgagctgt ccacctgcgg 1200 ctgcagccgc gccgcgcgcc ccaaggacct
gccgcgggac tggctctggg gcggctgcgg 1260 cgacaacatc gactatggct
accgctttgc caaggagttc gtggacgccc gcgagcggga 1320 gcgcatccac
gccaagggct cctacgagag tgctcgcatc ctcatgaacc tgcacaacaa 1380
cgaggccggc cgcaggacgg tgtacaacct ggctgatgtg gcctgcaagt gccatggggt
1440 gtccggctca tgtagcctga agacatgctg gctgcagctg gcagacttcc
gcaaggtggg 1500 tgatgccctg aaggagaagt acgacagcgc ggcggccatg
cggctcaaca gccggggcaa 1560 gttggtacag gtcaacagcc gcttcaactc
gcccaccaca caagacctgg tctacatcga 1620 ccccagccct gactactgcg
tgcgcaatga gagcaccggc tcgctgggca cgcagggccg 1680 cctgtgcaac
aagacgtcgg agggcatgga tggctgcgag ctcatgtgct gcggccgtgg 1740
gtacgaccag ttcaagaccg tgcagacgga gcgctgccac tgcaagttcc actggtgctg
1800 ctacgtcaag tgcaagaagt gcacggagat cgtggaccag tttgtgtgca
agtagtgggt 1860 gccacccagc actcagcccc gctcccagga cccgcttatt
tatagaaagt acagtgattc 1920 tggtttttgg tttttagaaa tattttttat
ttttccccaa gaattgcaac cggaaccatt 1980 ttttttcctg
ttaccatcta agaactctgt ggtttattat taatattata attattattt 2040
ggcaataatg ggggtgggaa ccacgaaaaa tatttatttt gtggatcttt gaaaaggtaa
2100 tacaagactt cttttggata gtatagaatg aagggggaaa taacacatac
cctaacttag 2160 ctgtgtggga catggtacac atccagaagg taaagaaata
cattttcttt ttctcaaata 2220 tgccatcata tgggatgggt aggttccagt
tgaaagaggg tggtagaaat ctattcacaa 2280 ttcagcttct atgaccaaaa
tgagttgtaa attctctggt gcaagataaa aggtcttggg 2340 aaaacaaaac
aaaacaaaac aaacctccct tccccagcag ggctgctagc ttgctttctg 2400
cattttcaaa atgataattt acaatggaag gacaagaatg tcatattctc aaggaaaaaa
2460 ggtatatcac atgtctcatt ctcctcaaat attccatttg cagacagacc
gtcatattct 2520 aatagctcat gaaatttggg cagcagggag gaaagtcccc
agaaattaaa aaatttaaaa 2580 ctcttatgtc aagatgttga tttgaagctg
ttataagaat tgggattcca gatttgtaaa 2640 aagaccccca atgattctgg
acactagatt ttttgtttgg ggaggttggc ttgaacataa 2700 atgaaatatc
ctgtattttc ttagggatac ttggttagta aattataata gtagaaataa 2760
tacatgaatc ccattcacag gtttctcagc ccaagcaaca aggtaattgc gtgccattca
2820 gcactgcacc agagcagaca acctatttga ggaaaaacag tgaaatccac
cttcctcttc 2880 acactgagcc ctctctgatt cctccgtgtt gtgatgtgat
gctggccacg tttccaaacg 2940 gcagctccac tgggtcccct ttggttgtag
gacaggaaat gaaacattag gagctctgct 3000 tggaaaacag ttcactactt
agggattttt gtttcctaaa acttttattt tgaggagcag 3060 tagttttcta
tgttttaatg acagaacttg gctaatggaa ttcacagagg tgttgcagcg 3120
tatcactgtt atgatcctgt gtttagatta tccactcatg cttctcctat tgtactgcag
3180 gtgtacctta aaactgttcc cagtgtactt gaacagttgc atttataagg
ggggaaatgt 3240 ggtttaatgg tgcctgatat ctcaaagtct tttgtacata
acatatatat atatatacat 3300 atatataaat ataaatataa atatatctca
ttgcagccag tgatttagat ttacagctta 3360 ctctggggtt atctctctgt
ctagagcatt gttgtccttc actgcagtcc agttgggatt 3420 attccaaaag
ttttttgagt cttgagcttg ggctgtggcc ccgctgtgat cataccctga 3480
gcacgacgaa gcaacctcgt ttctgaggaa gaagcttgag ttctgactca ctgaaatgcg
3540 tgttgggttg aagatatctt tttttctttt ctgcctcacc cctttgtctc
caacctccat 3600 ttctgttcac tttgtggaga gggcattact tgttcgttat
agacatggac gttaagagat 3660 attcaaaact cagaagcatc agcaatgttt
ctcttttctt agttcattct gcagaatgga 3720 aacccatgcc tattagaaat
gacagtactt attaattgag tccctaagga atattcagcc 3780 cactacatag
atagcttttt tttttttttt ttttttttaa taaggacacc tctttccaaa 3840
caggccatca aatatgttct tatctcagac ttacgttgtt ttaaaagttt ggaaagatac
3900 acatcttttc ataccccccc ttaggaggtt gggctttcat atcacctcag
ccaactgtgg 3960 ctcttaattt attgcataat gatatccaca tcagccaact
gtggctcttt aatttattgc 4020 ataatgatat tcacatcccc tcagttgcag
tgaattgtga gcaaaagatc ttgaaagcaa 4080 aaagcactaa ttagtttaaa
atgtcacttt tttggttttt attatacaaa aaccatgaag 4140 tacttttttt
atttgctaaa tcagattgtt cctttttagt gactcatgtt tatgaagaga 4200
gttgagttta acaatcctag cttttaaaag aaactattta atgtaaaata ttctacatgt
4260 cattcagata ttatgtatat cttctagcct ttattctgta cttttaatgt
acatatttct 4320 gtcttgcgtg atttgtatat ttcactggtt taaaaaacaa
acatcgaaag gcttattcca 4380 aatggaagat agaatataaa ataaaacgtt
acttgtaaaa aaaaaaaa 4428 15 359 PRT Homo sapiens human Wnt-5b 15
Met Pro Ser Leu Leu Leu Leu Phe Thr Ala Ala Leu Leu Ser Ser Trp 1 5
10 15 Ala Gln Leu Leu Thr Asp Ala Asn Ser Trp Trp Ser Leu Ala Leu
Asn 20 25 30 Pro Val Gln Arg Pro Glu Met Phe Ile Ile Gly Ala Gln
Pro Val Cys 35 40 45 Ser Gln Leu Pro Gly Leu Ser Pro Gly Gln Arg
Lys Leu Cys Gln Leu 50 55 60 Tyr Gln Glu His Met Ala Tyr Ile Gly
Glu Gly Ala Lys Thr Gly Ile 65 70 75 80 Lys Glu Cys Gln His Gln Phe
Arg Gln Arg Arg Trp Asn Cys Ser Thr 85 90 95 Ala Asp Asn Ala Ser
Val Phe Gly Arg Val Met Gln Ile Gly Ser Arg 100 105 110 Glu Thr Ala
Phe Thr His Ala Val Ser Ala Ala Gly Val Val Asn Ala 115 120 125 Ile
Ser Arg Ala Cys Arg Glu Gly Glu Leu Ser Thr Cys Gly Cys Ser 130 135
140 Arg Thr Ala Arg Pro Lys Asp Leu Pro Arg Asp Trp Leu Trp Gly Gly
145 150 155 160 Cys Gly Asp Asn Val Glu Tyr Gly Tyr Arg Phe Ala Lys
Glu Phe Val 165 170 175 Asp Ala Arg Glu Arg Glu Lys Asn Phe Ala Lys
Gly Ser Glu Glu Gln 180 185 190 Gly Arg Val Leu Met Asn Leu Gln Asn
Asn Glu Ala Gly Arg Arg Ala 195 200 205 Val Tyr Lys Met Ala Asp Val
Ala Cys Lys Cys His Gly Val Ser Gly 210 215 220 Ser Cys Ser Leu Lys
Thr Cys Trp Leu Gln Leu Ala Glu Phe Arg Lys 225 230 235 240 Val Gly
Asp Arg Leu Lys Glu Lys Tyr Asp Ser Ala Ala Ala Met Arg 245 250 255
Val Thr Arg Lys Gly Arg Leu Glu Leu Val Asn Ser Arg Phe Thr Gln 260
265 270 Pro Thr Pro Glu Asp Leu Val Tyr Val Asp Pro Ser Pro Asp Tyr
Cys 275 280 285 Leu Arg Asn Glu Ser Thr Gly Ser Leu Gly Thr Gln Gly
Arg Leu Cys 290 295 300 Asn Lys Thr Ser Glu Gly Met Asp Gly Cys Glu
Leu Met Cys Cys Gly 305 310 315 320 Arg Gly Tyr Asn Gln Phe Lys Ser
Val Gln Val Glu Arg Cys His Cys 325 330 335 Lys Phe His Trp Cys Cys
Phe Val Arg Cys Lys Lys Cys Thr Glu Ile 340 345 350 Val Asp Gln Tyr
Ile Cys Lys 355 16 2251 DNA Homo sapiens human Wnt-5b 16 gaccattagc
aggcacccag gcctgtcttt ggctcggaaa cggtggcccc caatgtagcc 60
tagtttgaac ctaggaactg caggaccaga gagattccac tggagcctga tggacgggtg
120 acagagggaa ccctactctg gaaactgtca gtcccagggc actggggagg
gctgaggccg 180 accatgccca gcctgctgct gctgttcacg gctgctctgc
tgtccagctg ggctcagctt 240 ctgacagacg ccaactcctg gtggtcatta
gctttgaacc cggtgcagag acccgagatg 300 tttatcatcg gtgcccagcc
cgtgtgcagt cagcttcccg ggctctcccc tggccagagg 360 aagctgtgcc
aattgtacca ggagcacatg gcctacatag gggagggagc caagactggc 420
atcaaggaat gccagcacca gttccggcag cggcggtgga attgcagcac agcggacaac
480 gcatctgtct ttgggagagt catgcagata ggcagccgag agaccgcctt
cacccacgcg 540 gtgagcgccg cgggcgtggt caacgccatc agccgggcct
gccgcgaggg cgagctctcc 600 acctgcggct gcagccggac ggcgcggccc
aaggacctgc cccgggactg gctgtggggc 660 ggctgtgggg acaacgtgga
gtacggctac cgcttcgcca aggagtttgt ggatgcccgg 720 gagcgagaga
agaactttgc caaaggatca gaggagcagg gccgggtgct catgaacctg 780
caaaacaacg aggccggtcg cagggctgtg tataagatgg cagacgtagc ctgcaaatgc
840 cacggcgtct cggggtcctg cagcctcaag acctgctggc tgcagctggc
cgagttccgc 900 aaggtcgggg accggctgaa ggagaagtac gacagcgcgg
ccgccatgcg cgtcacccgc 960 aagggccggc tggagctggt caacagccgc
ttcacccagc ccaccccgga ggacctggtc 1020 tatgtggacc ccagccccga
ctactgcctg cgcaacgaga gcacgggctc cctgggcacg 1080 cagggccgcc
tctgcaacaa gacctcggag ggcatggatg gctgtgagct catgtgctgc 1140
gggcgtggct acaaccagtt caagagcgtg caggtggagc gctgccactg caagttccac
1200 tggtgctgct tcgtcaggtg taagaagtgc acggagatcg tggaccagta
catctgtaaa 1260 tagcccggag ggcctgctcc cggcccccct gcactctgcc
tcacaaaggt ctatattata 1320 taaatctata taaatctatt ttatatttgt
ataagtaaat gggtgggtgc tatacaatgg 1380 aaagatgaaa atggaaagga
agagcttatt taagagacgc tggagatctc tgaggagtgg 1440 actttgctgg
ttctctcctc ttggtgggtg ggagacaggg ctttttctct ccctctggcg 1500
aggactctca ggatgtaggg acttggaaat atttactgtc tgtccaccac ggcctggagg
1560 agggaggttg tggttggatg gaggagatga tcttgtctgg aagtctagag
tctttgttgg 1620 ttagaggact gcctgtgatc ctggccacta ggccaagagg
ccctatgaag gtggcgggaa 1680 ctcagcttca acctcgatgt cttcagggtc
ttgtccagaa tgtagatggg ttccgtaaga 1740 ggcctggtgc tctcttactc
tttcatccac gtgcacttgt gcggcatctg cagtttacag 1800 gaacggctcc
ttccctaaaa tgagaagtcc aaggtcatct ctggcccagt gaccacagag 1860
agatctgcac ctcccggact tcaggcctgc ctttccagcg agaattcttc atcctccacg
1920 gttcactagc tcctacctga agaggaaagg gggccatttg acctgacatg
tcaggaaagc 1980 cctaaactga atgtttgcgc ctgggctgca gaagccaggg
tgcatgacca ggctgcgtgg 2040 acgttatact gtcttccccc acccccgggg
aggggaagct tgagctgctg ctgtcactcc 2100 tccaccgagg gaggcctcac
aaaccacagg acgctgcaac gggtcaggct ggcgggcccg 2160 gcgtgctcat
catctctgcc ccaggtgtac ggtttctctc tgacattaaa tgcccttcat 2220
ggaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 2251 17 365 PRT Homo sapiens
human Wnt-6 17 Met Leu Pro Pro Leu Pro Ser Arg Leu Gly Leu Leu Leu
Leu Leu Leu 1 5 10 15 Leu Cys Pro Ala His Val Gly Gly Leu Trp Trp
Ala Val Gly Ser Pro 20 25 30 Leu Val Met Asp Pro Thr Ser Ile Cys
Arg Lys Ala Arg Arg Leu Ala 35 40 45 Gly Arg Gln Ala Glu Leu Cys
Gln Ala Glu Pro Glu Val Val Ala Glu 50 55 60 Leu Ala Arg Gly Ala
Arg Leu Gly Val Arg Glu Cys Gln Phe Gln Phe 65 70 75 80 Arg Phe Arg
Arg Trp Asn Cys Ser Ser His Ser Lys Ala Phe Gly Arg 85 90 95 Ile
Leu Gln Gln Asp Ile Arg Glu Thr Ala Phe Val Phe Ala Ile Thr 100 105
110 Ala Ala Gly Ala Ser His Ala Val Thr Gln Ala Cys Ser Met Gly Glu
115 120 125 Leu Leu Gln Cys Gly Cys Gln Ala Pro Arg Gly Arg Ala Pro
Pro Arg 130 135 140 Pro Ser Gly Leu Pro Gly Thr Pro Gly Pro Pro Gly
Pro Ala Gly Ser 145 150 155 160 Pro Glu Gly Ser Ala Ala Trp Glu Trp
Gly Gly Cys Gly Asp Asp Val 165 170 175 Asp Phe Gly Asp Glu Lys Ser
Arg Leu Phe Met Asp Ala Arg His Lys 180 185 190 Arg Gly Arg Gly Asp
Ile Arg Ala Leu Val Gln Leu His Asn Asn Glu 195 200 205 Ala Gly Arg
Leu Ala Val Arg Ser His Thr Arg Thr Glu Cys Lys Cys 210 215 220 His
Gly Leu Ser Gly Ser Cys Ala Leu Arg Thr Cys Trp Gln Lys Leu 225 230
235 240 Pro Pro Phe Arg Glu Val Gly Ala Arg Leu Leu Glu Arg Phe His
Gly 245 250 255 Ala Ser Arg Val Met Gly Thr Asn Asp Gly Lys Ala Leu
Leu Pro Ala 260 265 270 Val Arg Thr Leu Lys Pro Pro Gly Arg Ala Asp
Leu Leu Tyr Ala Ala 275 280 285 Asp Ser Pro Asp Phe Cys Ala Pro Asn
Arg Arg Thr Gly Ser Pro Gly 290 295 300 Thr Arg Gly Arg Ala Cys Asn
Ser Ser Ala Pro Asp Leu Ser Gly Cys 305 310 315 320 Asp Leu Leu Cys
Cys Gly Arg Gly His Arg Gln Glu Ser Val Gln Leu 325 330 335 Glu Glu
Asn Cys Leu Cys Arg Phe His Trp Cys Cys Val Val Gln Cys 340 345 350
His Arg Cys Arg Val Arg Lys Glu Leu Ser Leu Cys Leu 355 360 365 18
1726 DNA Homo sapiens human Wnt-6 18 ggcacgagcg caggagacac
aggcgctggc tgccccgtcc gctctccgcc tccgccgcgc 60 cctcctcgcc
cgggatgggc ccccccgccg ccgccggatc cctcgcctcc cggccgccgc 120
cgttgcgctc gccgcgctcg cactgaagcc cgggccctcg cgcgccgcgg ttcgccccgc
180 agcctcgccc cctgcccacc cgggcggccg tagggcggtc acgatgctgc
cgcccttacc 240 ctcccgcctc gggctgctgc tgctgctgct cctgtgcccg
gcgcacgtcg gcggactgtg 300 gtgggctgtg ggcagcccct tggttatgga
ccctaccagc atctgcagga aggcacggcg 360 gctggccggg cggcaggccg
agttgtgcca ggctgagccg gaagtggtgg cagagctagc 420 tcggggcgcc
cggctcgggg tgcgagagtg ccagttccag ttccgcttcc gccgctggaa 480
ttgctccagc cacagcaagg cctttggacg catcctgcaa caggacattc gggagacggc
540 cttcgtgttc gccatcactg cggccggcgc cagccacgcc gtcacgcagg
cctgttctat 600 gggcgagctg ctgcagtgcg gctgccaggc gccccgcggg
cgggcccctc cccggccctc 660 cggcctgccc ggcacccccg gaccccctgg
ccccgcgggc tccccggaag gcagcgccgc 720 ctgggagtgg ggaggctgcg
gcgacgacgt ggacttcggg gacgagaagt cgaggctctt 780 tatggacgcg
cggcacaagc ggggacgcgg agacatccgc gcgttggtgc aactgcacaa 840
caacgaggcg ggcaggctgg ccgtgcggag ccacacgcgc accgagtgca aatgccacgg
900 gctgtcggga tcatgcgcgc tgcgcacctg ctggcagaag ctgcctccat
ttcgcgaggt 960 gggcgcgcgg ctgctggagc gcttccacgg cgcctcacgc
gtcatgggca ccaacgacgg 1020 caaggccctg ctgcccgccg tccgcacgct
caagccgccg ggccgagcgg acctcctcta 1080 cgccgccgat tcgcccgact
tttgcgcccc caaccgacgc accggctccc ccggcacgcg 1140 cggtcgcgcc
tgcaatagca gcgccccgga cctcagcggc tgcgacctgc tgtgctgcgg 1200
ccgcgggcac cgccaggaga gcgtgcagct cgaagagaac tgcctgtgcc gcttccactg
1260 gtgctgcgta gtacagtgcc accgttgccg tgtgcgcaag gagctcagcc
tctgcctgtg 1320 acccgccgcc cggccgctag actgacttcg cgcagcggtg
gctcgcacct gtgggacctc 1380 agggcaccgg caccgggcgc ctctcgccgc
tcgagcccag cctctccctg ccaaagccca 1440 actcccaggg ctctggaaat
ggtgaggcga ggggcttgag aggaacgccc acccacgaag 1500 gcccagggcg
ccagacggcc ccgaaaaggc gctcggggag cgtttaaagg acactgtaca 1560
ggccctccct ccccttggcc tctaggagga aacagttttt tagactggaa aaaagccagt
1620 ctaaaggcct ctggatactg ggctccccag aactgctggc cacaggatgg
tgggtgaggt 1680 tagtatcaat aaagatattt aaaccaaaaa aaaaaaaaaa aaaaaa
1726 19 349 PRT Homo sapiens human Wnt-7a 19 Met Asn Arg Lys Ala
Arg Arg Cys Leu Gly His Leu Phe Leu Ser Leu 1 5 10 15 Gly Met Val
Tyr Leu Arg Ile Gly Gly Phe Ser Ser Val Val Ala Leu 20 25 30 Gly
Ala Ser Ile Ile Cys Asn Lys Ile Pro Gly Leu Ala Pro Arg Gln 35 40
45 Arg Ala Ile Cys Gln Ser Arg Pro Asp Ala Ile Ile Val Ile Gly Glu
50 55 60 Gly Ser Gln Met Gly Leu Asp Glu Cys Gln Phe Gln Phe Arg
Asn Gly 65 70 75 80 Arg Trp Asn Cys Ser Ala Leu Gly Glu Arg Thr Val
Phe Gly Lys Glu 85 90 95 Leu Lys Val Gly Ser Arg Glu Ala Ala Phe
Thr Tyr Ala Ile Ile Ala 100 105 110 Ala Gly Val Ala His Ala Ile Thr
Ala Ala Cys Thr Gln Gly Asn Leu 115 120 125 Ser Asp Cys Gly Cys Asp
Lys Glu Lys Gln Gly Gln Tyr His Arg Asp 130 135 140 Glu Gly Trp Lys
Trp Gly Gly Cys Ser Ala Asp Ile Arg Tyr Gly Ile 145 150 155 160 Gly
Phe Ala Lys Val Phe Val Asp Ala Arg Glu Ile Lys Gln Asn Ala 165 170
175 Arg Thr Leu Met Asn Leu His Asn Asn Glu Ala Gly Arg Lys Ile Leu
180 185 190 Glu Glu Asn Met Lys Leu Glu Cys Lys Cys His Gly Val Ser
Gly Ser 195 200 205 Cys Thr Thr Lys Thr Cys Trp Thr Thr Leu Pro Gln
Phe Arg Glu Leu 210 215 220 Gly Tyr Val Leu Lys Asp Lys Tyr Asn Glu
Ala Val His Val Glu Pro 225 230 235 240 Val Arg Ala Ser Arg Asn Lys
Arg Pro Thr Phe Leu Lys Ile Lys Lys 245 250 255 Pro Leu Ser Tyr Arg
Lys Pro Met Asp Thr Asp Leu Val Tyr Ile Glu 260 265 270 Lys Ser Pro
Asn Tyr Cys Glu Glu Asp Pro Val Thr Gly Ser Val Gly 275 280 285 Thr
Gln Gly Arg Ala Cys Asn Lys Thr Ala Pro Gln Ala Ser Gly Cys 290 295
300 Asp Leu Met Cys Cys Gly Arg Gly Tyr Asn Thr His Gln Tyr Ala Arg
305 310 315 320 Val Trp Gln Cys Asn Cys Lys Phe His Trp Cys Cys Tyr
Val Lys Cys 325 330 335 Asn Thr Cys Ser Glu Arg Thr Glu Met Tyr Thr
Cys Lys 340 345 20 1736 DNA Homo sapiens human Wnt-7a 20 gagggagggg
cgggggctgg aggcagcagc gcccccgcac tccccgcgtc tcgcacactt 60
gcaccggtcg ctcgcgcgca gcccggcgtc gccccacgcc gcgctcgctc ctccctccct
120 cctcccgctc cgtggctccc gtgctcctgg cgaggctcag gcgcggagcg
cgcggacggg 180 cgcaccgaca gacggccccg gggacgcctc ggctcgcgcc
tcccgggcgg gctatgttga 240 ttgccccgcc ggggccggcc cgcgggatca
gcacagcccg gcccgcggcc ccggcggcca 300 atcgggacta tgaaccggaa
agcgcggcgc tgcctgggcc acctctttct cagcctgggc 360 atggtctacc
tccggatcgg tggcttctcc tcagtggtag ctctgggcgc aagcatcatc 420
tgtaacaaga tcccaggcct ggctcccaga cagcgggcga tctgccagag ccggcccgac
480 gccatcatcg tcataggaga aggctcacaa atgggcctgg acgagtgtca
gtttcagttc 540 cgcaatggcc gctggaactg ctctgcactg ggagagcgca
ccgtcttcgg gaaggagctc 600 aaagtgggga gccgggaggc tgcgttcacc
tacgccatca ttgccgccgg cgtggcccac 660 gccatcacag ctgcctgtac
ccagggcaac ctgagcgact gtggctgcga caaagagaag 720 caaggccagt
accaccggga cgagggctgg aagtggggtg gctgctctgc cgacatccgc 780
tacggcatcg gcttcgccaa ggtctttgtg gatgcccggg agatcaagca gaatgcccgg
840 actctcatga acttgcacaa caacgaggca ggccgaaaga tcctggagga
gaacatgaag 900 ctggaatgta agtgccacgg cgtgtcaggc tcgtgcacca
ccaagacgtg ctggaccaca 960 ctgccacagt ttcgggagct gggctacgtg
ctcaaggaca agtacaacga ggccgttcac 1020 gtggagcctg tgcgtgccag
ccgcaacaag cggcccacct tcctgaagat caagaagcca 1080 ctgtcgtacc
gcaagcccat ggacacggac ctggtgtaca tcgagaagtc gcccaactac 1140
tgcgaggagg acccggtgac cggcagtgtg ggcacccagg gccgcgcctg caacaagacg
1200 gctccccagg ccagcggctg tgacctcatg tgctgtgggc gtggctacaa
cacccaccag 1260 tacgcccgcg tgtggcagtg caactgtaag ttccactggt
gctgctatgt caagtgcaac 1320 acgtgcagcg agcgcacgga gatgtacacg
tgcaagtgag ccccgtgtgc acaccaccct 1380 cccgctgcaa gtcagattgc
tgggaggact ggaccgtttc caagctgcgg gctccctggc 1440 aggatgctga
gcttgtcttt tctgctgagg agggtacttt tcctgggttt cctgcaggca 1500
tccgtggggg aaaaaaaatc tctcagagcc ctcaactatt ctgttccaca cccaatgctg
1560 ctccaccctc ccccagacac agcccaggtc cctccgcggc tggagcgaag
ccttctgcag 1620 caggaactct ggacccctgg gcctcatcac agcaatattt
aacaatttat tctgataaaa 1680 ataatattaa tttatttaat taaaaagaat
tcttccacaa aaaaaaaaaa aaaaaa 1736 21 349 PRT Homo sapiens human
Wnt-7b 21 Met His Arg
Asn Phe Arg Lys Trp Ile Phe Tyr Val Phe Leu Cys Phe 1 5 10 15 Gly
Val Leu Tyr Val Lys Leu Gly Ala Leu Ser Ser Val Val Ala Leu 20 25
30 Gly Ala Asn Ile Ile Cys Asn Lys Ile Pro Gly Leu Ala Pro Arg Gln
35 40 45 Arg Ala Ile Cys Gln Ser Arg Pro Asp Ala Ile Ile Val Ile
Gly Glu 50 55 60 Gly Ala Gln Met Gly Ile Asn Glu Cys Gln Tyr Gln
Phe Arg Phe Gly 65 70 75 80 Arg Trp Asn Cys Ser Ala Leu Gly Glu Lys
Thr Val Phe Gly Gln Glu 85 90 95 Leu Arg Val Gly Ser Arg Glu Ala
Ala Phe Thr Tyr Ala Ile Thr Ala 100 105 110 Ala Gly Val Ala His Ala
Val Thr Ala Ala Cys Ser Gln Gly Asn Leu 115 120 125 Ser Asn Cys Gly
Cys Asp Arg Glu Lys Gln Gly Tyr Tyr Asn Gln Ala 130 135 140 Glu Gly
Trp Lys Trp Gly Gly Cys Ser Ala Asp Val Arg Tyr Gly Ile 145 150 155
160 Asp Phe Ser Arg Arg Phe Val Asp Ala Arg Glu Ile Lys Lys Asn Ala
165 170 175 Arg Arg Leu Met Asn Leu His Asn Asn Glu Ala Gly Arg Lys
Val Leu 180 185 190 Glu Asp Arg Met Gln Leu Glu Cys Lys Cys His Gly
Val Ser Gly Ser 195 200 205 Cys Thr Thr Lys Thr Cys Trp Thr Thr Leu
Pro Lys Phe Arg Glu Val 210 215 220 Gly His Leu Leu Lys Glu Lys Tyr
Asn Ala Ala Val Gln Val Glu Val 225 230 235 240 Val Arg Ala Ser Arg
Leu Arg Gln Pro Thr Phe Leu Arg Ile Lys Gln 245 250 255 Leu Arg Ser
Tyr Gln Lys Pro Met Glu Thr Asp Leu Val Tyr Ile Glu 260 265 270 Lys
Ser Pro Asn Tyr Cys Glu Glu Asp Ala Ala Thr Gly Ser Val Gly 275 280
285 Thr Gln Gly Arg Leu Cys Asn Arg Thr Ser Pro Gly Ala Asp Gly Cys
290 295 300 Asp Thr Met Cys Cys Gly Arg Gly Tyr Asn Thr His Gln Tyr
Thr Lys 305 310 315 320 Val Trp Gln Cys Asn Cys Lys Phe His Trp Cys
Cys Phe Val Lys Cys 325 330 335 Asn Thr Cys Ser Glu Arg Thr Glu Val
Phe Thr Cys Lys 340 345 22 2250 DNA Homo sapiens human Wnt-7b 22
gagtctgccc gcagccccct ggcccctgcc cggccctgcg tgcccgcgcg tccctccggc
60 cgcgctgtct atggcgcagc ccccctccct ggatcatgca cagaaacttt
cgcaagtgga 120 ttttctacgt gtttctctgc tttggcgtcc tgtacgtgaa
gctcggagca ctgtcatccg 180 tggtggccct gggagccaac atcatctgca
acaagattcc tggcctagcc ccgcggcagc 240 gtgccatctg ccagagtcgg
cccgatgcca tcattgtgat tggggagggg gcgcagatgg 300 gcatcaacga
gtgccagtac cagttccgct tcggacgctg gaactgctct gccctcggcg 360
agaagaccgt cttcgggcaa gagctccgag tagggagccg tgaggctgcc ttcacgtacg
420 ccatcaccgc ggctggcgtg gcgcacgccg tcaccgctgc ctgcagccaa
gggaacctga 480 gcaactgcgg ctgcgaccgc gagaagcagg gctactacaa
ccaagccgag ggctggaagt 540 ggggcggctg ctcggccgac gtgcgttacg
gcatcgactt ctcccggcgc ttcgtggacg 600 ctcgggagat caagaagaac
gcgcggcgcc tcatgaacct gcataacaat gaggccggca 660 ggaaggttct
agaggaccgg atgcagctgg agtgcaagtg ccacggcgtg tctggctcct 720
gcaccaccaa aacctgctgg accacgctgc ccaagttccg agaggtgggc cacctgctga
780 aggagaagta caacgcggcc gtgcaggtgg aggtggtgcg ggccagccgt
ctgcggcagc 840 ccaccttcct gcgcatcaaa cagctgcgca gctatcagaa
gcccatggag acagacctgg 900 tgtacattga gaagtcgccc aactactgcg
aggaggacgc ggccacgggc agcgtgggca 960 cgcagggccg tctctgcaac
cgcacgtcgc ccggcgcgga cggctgtgac accatgtgct 1020 gcggccgagg
ctacaacacc caccagtaca ccaaggtgtg gcagtgcaac tgcaaattcc 1080
actggtgctg cttcgtcaag tgcaacacct gcagcgagcg caccgaggtc ttcacctgca
1140 agtgaggcca ggcccggagg cggccgcggg caccctggaa cccggcggca
ttttgcacat 1200 ccactcctca ccttccctgc cttggtgctg ccagcagcag
acatagacgg gtgcagaagc 1260 ggggagctcc aggtgcagga gggcaccggc
cggggcccac gccctctgcc cgcctccctg 1320 gggctccttc ctgccacctc
ctcccatcac ctcctgcggc agaacagcac ccgtgaccca 1380 cccagagagc
aaggccaggg gtcttggtgc tccccgacgg ggcccggcaa gttctctttc 1440
ttctctctgg gaaaatgaac gtccaggaca cacctgtatc ccagagagca aagtgatgag
1500 gagactgagc gtccccagcc ccacctggcg gcatggacac agaaaagcta
cgccggctgg 1560 cctctccaga ccagttccca ggctgggtct gccgctgggc
cctggggcgg tggggacaga 1620 tgttgacaca aattatttat gttttcttag
tatcagaaga ggattctcgg cactaacaca 1680 tagccagtcc taactccgta
ctctgtgtca gcccatcccc tagacaccct ctgtttcctt 1740 tcccggcccc
acctggccgg ccctctgccc ctgcagagct gaggcagcct ggggttgatg 1800
gggaccacgc ggtgcctgca ggtcctagaa gtgagctggg caggggctct tcagaccaca
1860 cagccctgac cgggccttgg aggagagcca tggacaggct cctccatgcc
gtctttcctt 1920 cttttgaaaa tcctatcaat ggctgggcgc ggtggctcac
acctgtaatc ccagcacttt 1980 gggagaccga ggcaggtgga tcacctgagg
tcaggagttc gagaccagcc tggccaacgt 2040 ggtgaaaccc tgtctctact
aaaaatacaa aaattagctg ggcgtggtgg cgtgcacctg 2100 taatcccagc
tactcaggag gctgagacag gacacttgct tgaacccggg aggtggaggt 2160
tgcaatgagc caagattgtg ccactgtatt ccaacttggg tgacagagca cgactctgtc
2220 tcaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2250 23 355 PRT Homo sapiens
human Wnt-8a 23 Met Gly Asn Leu Phe Met Leu Trp Ala Ala Leu Gly Ile
Cys Cys Ala 1 5 10 15 Ala Phe Ser Ala Ser Ala Trp Ser Val Asn Asn
Phe Leu Ile Thr Gly 20 25 30 Pro Lys Ala Tyr Leu Thr Tyr Thr Thr
Ser Val Ala Leu Gly Ala Gln 35 40 45 Ser Gly Ile Glu Glu Cys Lys
Phe Gln Phe Ala Trp Glu Arg Trp Asn 50 55 60 Cys Pro Glu Asn Ala
Leu Gln Leu Ser Thr His Asn Arg Leu Arg Ser 65 70 75 80 Ala Thr Arg
Glu Thr Ser Phe Ile His Ala Ile Ser Ser Ala Gly Val 85 90 95 Met
Tyr Ile Ile Thr Lys Asn Cys Ser Met Gly Asp Phe Glu Asn Cys 100 105
110 Gly Cys Asp Gly Ser Asn Asn Gly Lys Thr Gly Gly His Gly Trp Ile
115 120 125 Trp Gly Gly Cys Ser Asp Asn Val Glu Phe Gly Glu Arg Ile
Ser Lys 130 135 140 Leu Phe Val Asp Ser Leu Glu Lys Gly Lys Asp Ala
Arg Ala Leu Met 145 150 155 160 Asn Leu His Asn Asn Arg Ala Gly Arg
Leu Ala Val Arg Ala Thr Met 165 170 175 Lys Arg Thr Cys Lys Cys His
Gly Ile Ser Gly Ser Cys Ser Ile Gln 180 185 190 Thr Cys Trp Leu Gln
Leu Ala Glu Phe Arg Glu Met Gly Asp Tyr Leu 195 200 205 Lys Ala Lys
Tyr Asp Gln Ala Leu Lys Ile Glu Met Asp Lys Arg Gln 210 215 220 Leu
Arg Ala Gly Asn Ser Ala Glu Gly His Trp Val Pro Ala Glu Ala 225 230
235 240 Phe Leu Pro Ser Ala Glu Ala Glu Leu Ile Phe Leu Glu Glu Ser
Pro 245 250 255 Asp Tyr Cys Thr Cys Asn Ser Ser Leu Gly Ile Tyr Gly
Thr Glu Gly 260 265 270 Arg Glu Cys Leu Gln Asn Ser His Asn Thr Ser
Arg Trp Glu Arg Arg 275 280 285 Ser Cys Gly Arg Leu Cys Thr Glu Cys
Gly Leu Gln Val Glu Glu Arg 290 295 300 Lys Thr Glu Val Ile Ser Ser
Cys Asn Cys Lys Phe Gln Trp Cys Cys 305 310 315 320 Thr Val Lys Cys
Asp Gln Cys Arg His Val Val Ser Lys Tyr Tyr Cys 325 330 335 Ala Arg
Ser Pro Gly Ser Ala Gln Ser Leu Gly Arg Val Trp Phe Gly 340 345 350
Val Tyr Ile 355 24 1597 DNA Homo sapiens human Wnt-8a 24 cagaattttc
tcacataaat actgaggaag accctgccct ctcctcactc ctctggactt 60
ggccctgagc tggacctggt ccactggggt aggcagggcg atggggaacc tgtttatgct
120 ctgggcagct ctgggcatat gctgtgctgc attcagtgcc tctgcctggt
cagtgaacaa 180 tttcctgata acaggtccca aggcctatct gacctacacg
actagtgtgg ccttgggtgc 240 ccagagtggc atcgaggagt gcaagttcca
gtttgcttgg gaacgctgga actgccctga 300 aaatgctctt cagctctcca
cccacaacag gctgagaagt gctaccagag agacttcctt 360 catacatgct
atcagctctg ctggagtcat gtacatcatc accaagaact gtagcatggg 420
tgacttcgaa aactgtggct gtgatgggtc aaacaatgga aaaacaggag gccatggctg
480 gatctgggga ggctgcagcg acaatgtgga atttggggaa aggatctcca
aactctttgt 540 ggacagtttg gagaagggga aggatgccag agccctgatg
aatcttcaca acaacagggc 600 cggcagactg gcagtgagag ccaccatgaa
aaggacatgc aaatgtcatg gcatctctgg 660 gagctgcagc atacagacat
gctggctgca gctggctgaa ttccgggaga tgggagacta 720 cctaaaggcc
aagtatgacc aggcgctgaa aattgaaatg gataagcggc agctgagagc 780
tgggaacagc gccgagggcc actgggtgcc cgctgaggcc ttccttccta gcgcagaggc
840 ggaactgatc tttttagagg aatcaccaga ttactgtacc tgcaattcca
gcctgggcat 900 ctatggcaca gagggtcgtg agtgcctaca gaacagccac
aacacatcca ggtgggagcg 960 acgtagctgt gggcgcctgt gcactgagtg
tgggctgcag gtggaagaga ggaaaactga 1020 ggtcataagc agctgtaact
gcaaattcca gtggtgctgt acggtcaagt gtgaccagtg 1080 taggcatgtg
gtgagcaagt attactgcgc acgctcccca ggcagtgccc agtccctggg 1140
gagagtttgg tttggggtct atatctagag ggaccttcaa agtatttgtt cctttaaatt
1200 tcagaccatg tccaacccag ctgtgctgct gggaatcagg agaatagaag
caaaaaacga 1260 aagagttctg ttcagacttc tgaagagcag cctgtggcta
caaatctatg ctgataaatg 1320 agattgagaa ctcaactgta ttttgccata
aatgcttcta agatatatcc agctgggact 1380 tctattactc cctttggaaa
ccttaagatc aaaaagggaa taagaaaccc ttcttctgta 1440 tcccaataat
ccaccaggat aaaggagaaa ctagaaatat gcaactccct tgatttcagt 1500
gtttggcagg taacaaaaaa ttgagaccca gacactggtc aacaggaaaa caatacagac
1560 tcccagaatt agaaagtgtt attttaatgc aacctag 1597 25 351 PRT Homo
sapiens human Wnt-8b 25 Met Phe Leu Ser Lys Pro Ser Val Tyr Ile Cys
Leu Phe Thr Cys Val 1 5 10 15 Leu Gln Leu Ser His Ser Trp Ser Val
Asn Asn Phe Leu Met Thr Gly 20 25 30 Pro Lys Ala Tyr Leu Ile Tyr
Ser Ser Ser Val Ala Ala Gly Ala Gln 35 40 45 Ser Gly Ile Glu Glu
Cys Lys Tyr Gln Phe Ala Trp Asp Arg Trp Asn 50 55 60 Cys Pro Glu
Arg Ala Leu Gln Leu Ser Ser His Gly Gly Leu Arg Ser 65 70 75 80 Ala
Asn Arg Glu Thr Ala Phe Val His Ala Ile Ser Ser Ala Gly Val 85 90
95 Met Tyr Thr Leu Thr Arg Asn Cys Ser Leu Gly Asp Phe Asp Asn Cys
100 105 110 Gly Cys Asp Asp Ser Arg Asn Gly Gln Leu Gly Gly Gln Gly
Trp Leu 115 120 125 Trp Gly Gly Cys Ser Asp Asn Val Gly Phe Gly Glu
Ala Ile Ser Lys 130 135 140 Gln Phe Val Asp Ala Leu Glu Thr Gly Gln
Asp Ala Arg Ala Ala Met 145 150 155 160 Asn Leu His Asn Asn Glu Ala
Gly Arg Lys Ala Val Lys Gly Thr Met 165 170 175 Lys Arg Thr Cys Lys
Cys His Gly Val Ser Gly Ser Cys Thr Thr Gln 180 185 190 Thr Cys Trp
Leu Gln Leu Pro Glu Phe Arg Glu Val Gly Ala His Leu 195 200 205 Lys
Glu Lys Tyr His Ala Ala Leu Lys Val Asp Leu Leu Gln Gly Ala 210 215
220 Gly Asn Ser Ala Ala Ala Arg Gly Ala Ile Ala Asp Thr Phe Arg Ser
225 230 235 240 Ile Ser Thr Arg Glu Leu Val His Leu Glu Asp Ser Pro
Asp Tyr Cys 245 250 255 Leu Glu Asn Lys Thr Leu Gly Leu Leu Gly Thr
Glu Gly Arg Glu Cys 260 265 270 Leu Arg Arg Gly Arg Ala Leu Gly Arg
Trp Glu Leu Arg Ser Cys Arg 275 280 285 Arg Leu Cys Gly Asp Cys Gly
Leu Ala Val Glu Glu Arg Arg Ala Glu 290 295 300 Thr Val Ser Ser Cys
Asn Cys Lys Phe His Trp Cys Cys Ala Val Arg 305 310 315 320 Cys Glu
Gln Cys Arg Arg Arg Val Thr Lys Tyr Phe Cys Ser Arg Ala 325 330 335
Glu Arg Pro Arg Gly Gly Ala Ala His Lys Pro Gly Arg Lys Pro 340 345
350 26 2117 DNA Homo sapiens human Wnt-8b 26 tccgcttaca caccaaggaa
agttgggctt tgaagaattc catccccatg gccactggag 60 gaagaatatt
tctccgtctt gcttacccat ctcccagttt tttggaattt tctctagctg 120
ttactccaga ggattatgtt tctttcaaag ccttctgtgt acatctgtct tttcacctgt
180 gtcctccaac tcagccacag ctggtcggtg aacaatttcc tgatgactgg
tccaaaggct 240 tacctgattt actccagcag tgtggcagct ggtgcccaga
gtggtattga agaatgcaag 300 tatcagtttg cctgggaccg ctggaactgc
cctgagagag ccctgcagct gtccagccat 360 ggtgggcttc gcagtgccaa
tcgggagaca gcatttgtgc atgccatcag ttctgctgga 420 gtcatgtaca
ccctgactag aaactgcagc cttggagatt ttgataactg tggctgtgat 480
gactcccgca acgggcaact ggggggacaa ggctggctgt ggggaggctg cagtgacaat
540 gtgggcttcg gagaggcgat ttccaagcag tttgtcgatg ccctggaaac
aggacaggat 600 gcacgggcag ccatgaacct gcacaacaac gaggctggcc
gcaaggcggt gaagggcacc 660 atgaaacgca cgtgtaagtg ccatggcgtg
tctggcagct gcaccacgca gacctgttgg 720 ctgcagctgc ccgagttccg
cgaggtgggc gcgcacctga aggagaagta ccacgcagca 780 ctcaaggtgg
acctgctgca gggtgctggc aacagcgcgg ccgcccgcgg cgccatcgcc 840
gacacctttc gctccatctc tacccgggag ctggtgcacc tggaggactc cccggactac
900 tgcctggaga acaaaacgct agggctgctg ggcaccgaag gccgagagtg
cctaaggcgc 960 gggcgggccc tgggtcgctg ggaactccgc agctgccgcc
ggctctgcgg ggactgcggg 1020 ctggcggtgg aggagcgccg ggccgagacc
gtgtccagct gcaactgcaa gttccactgg 1080 tgctgtgcag tccgctgcga
gcagtgccgc cggagggtca ccaagtactt ctgtagccgc 1140 gcagagcggc
cgcggggggg cgctgcgcac aaacccggga gaaaacccta agggtttcct 1200
ctgccccctc cttttcccac tggttcttgg cttcctttag agaccccggt aattgtggaa
1260 cctagggaat ggggaacccg ctctcccaga cctagggatc ctgaaaggga
aaaactgcaa 1320 tttctccaaa gcttgccact ttccagcctg tttccccaat
tcctctgtgc tctcctaaag 1380 ctctgtctga atcctcgcag ccacacctag
gtctgaaaac tcaggctttg agttactgat 1440 cttccttgga ttaggaaaac
aggtgttcct cctcccctct cctatcagcc ctaatctctg 1500 acctagccta
tcaaccctta ggcgctggaa aaaccttctc atacacgcag gacccaggtt 1560
aactcaaagc tttgcccttt tgcccactgt ctgctaccag gggctcaccc tctgctgcac
1620 ctctcttctg cacagctcct cccctgctac tgctgaccaa attcccagga
atcttgaatg 1680 ctttctctcc tcttctccct ttcctttccc aaaaaaaact
gaggaaactg gccccggaaa 1740 agcatgtctt tggggttggt tcctagaggc
agaggttgaa gatggaagag ggagctctgg 1800 agtgctaact tgaacaccaa
gggtgctact catccctatg gtatcatatc atgaatggac 1860 tttactagtg
gggcaatgac tttcctagac aataacccga gggactccag atacataccc 1920
cgaaggtcta ggaaatacgt taagggcaga ttacagtcat ttcctaccct ttaaaggtaa
1980 cttctccctt ctcctgacct acttcctcct agcaaccaac tttacctctt
cttctccaaa 2040 ggatctttgt tcctctgagc caagactgag gtaaataaag
ccactttcct cttcagatcc 2100 tggtctgcac ctctaga 2117 27 417 PRT Homo
sapiens human Wnt-10a 27 Met Gly Ser Ala His Pro Arg Pro Trp Leu
Arg Leu Arg Pro Gln Pro 1 5 10 15 Gln Pro Arg Pro Ala Leu Trp Val
Leu Leu Phe Phe Leu Leu Leu Leu 20 25 30 Ala Ala Ala Met Pro Arg
Ser Ala Pro Asn Asp Ile Leu Asp Leu Arg 35 40 45 Leu Pro Pro Glu
Pro Val Leu Asn Ala Asn Thr Val Cys Leu Thr Leu 50 55 60 Pro Gly
Leu Ser Arg Arg Gln Met Glu Val Cys Val Arg His Pro Asp 65 70 75 80
Val Ala Ala Ser Ala Ile Gln Gly Ile Gln Ile Ala Ile His Glu Cys 85
90 95 Gln His Gln Phe Arg Asp Gln Arg Trp Asn Cys Ser Ser Leu Glu
Thr 100 105 110 Arg Asn Lys Ile Pro Tyr Glu Ser Pro Ile Phe Ser Arg
Gly Phe Arg 115 120 125 Glu Ser Ala Phe Ala Tyr Ala Ile Ala Ala Ala
Gly Val Val His Ala 130 135 140 Val Ser Asn Ala Cys Ala Leu Gly Lys
Leu Lys Ala Cys Gly Cys Asp 145 150 155 160 Ala Ser Arg Arg Gly Asp
Glu Glu Ala Phe Arg Arg Lys Leu His Arg 165 170 175 Leu Gln Leu Asp
Ala Leu Gln Arg Gly Lys Gly Leu Ser His Gly Val 180 185 190 Pro Glu
His Pro Ala Leu Pro Thr Ala Ser Pro Gly Leu Gln Asp Ser 195 200 205
Trp Glu Trp Gly Gly Cys Ser Pro Asp Met Gly Phe Gly Glu Arg Phe 210
215 220 Ser Lys Asp Phe Leu Asp Ser Arg Glu Pro His Arg Asp Ile His
Ala 225 230 235 240 Arg Met Arg Leu His Asn Asn Arg Val Gly Arg Gln
Ala Val Met Glu 245 250 255 Asn Met Arg Arg Lys Cys Lys Cys His Gly
Thr Ser Gly Ser Cys Gln 260 265 270 Leu Lys Thr Cys Trp Gln Val Thr
Pro Glu Phe Arg Thr Val Gly Ala 275 280 285 Leu Leu Arg Ser Arg Phe
His Arg Ala Thr Leu Ile Arg Pro His Asn 290 295 300 Arg Asn Gly Gly
Gln Leu Glu Pro Gly Pro Ala Gly Ala Pro Ser Pro 305 310 315 320 Ala
Pro Gly Ala Pro Gly Pro Arg Arg Arg Ala Ser Pro Ala Asp Leu 325 330
335 Val Tyr Phe Glu Lys Ser Pro Asp Phe Cys Glu Arg Glu Pro Arg Leu
340 345 350 Asp Ser Ala Gly Thr Val Gly Arg Leu Cys Asn Lys Ser Ser
Ala Gly 355 360 365 Ser Asp Gly Cys Gly Ser Met Cys Cys Gly Arg Gly
His Asn Ile
Leu 370 375 380 Arg Gln Thr Arg Ser Glu Arg Cys His Cys Arg Phe His
Trp Cys Cys 385 390 395 400 Phe Val Val Cys Glu Glu Cys Arg Ile Thr
Glu Trp Val Ser Val Cys 405 410 415 Lys 28 2375 DNA Homo sapiens
human Wnt-10a 28 acagtcactt actctacagg cagtggggcc cgacacagac
agcgccgccc ccgccagcca 60 gcctcgcacg ccctcggaag cgcaggctcc
cggcgctgcg ctggagggtt ccccggcacc 120 ccagcctccc gtccccagcc
cgctgcacct ccgggccccc cttacccttg agaggcaccg 180 ggagttgtcg
cgggggggcc tcgggaaatt ccccggaccc ctgtgccagg aggtgcccgg 240
ttcgcccgct cttcaccccc cgcccccccc gagggcggtg cccgggggtg ctgccccatg
300 gagcggggag gcgggcgccg tctgctccgg gagccctgac ccgagtcgga
gctgtgtgtc 360 gcagccgccc cgaccccccg ccgatcatgc gccggcgccc
ctggctctcc agtcccactg 420 ggctgtgagc cccccactcc cagcccgtca
gggcctgcgc gccatgggca gcgcccaccc 480 tcgcccctgg ctgcggctcc
gaccccagcc ccagccgcgg ccagcgctct gggtgctcct 540 gttcttccta
ctgctgctgg ctgctgccat gcccaggtca gcacccaatg acattctgga 600
cctccgcctc cccccggagc ccgtgctcaa tgccaacaca gtgtgcctaa cattgccagg
660 cctgagccgg cggcagatgg aggtgtgtgt gcgtcaccct gatgtggctg
cctcagccat 720 acagggcatc cagatcgcca tccacgaatg ccaacaccaa
ttcagggacc agcgctggaa 780 ctgctcaagc ctggagactc gcaacaagat
cccctatgag agtcccatct tcagcagagg 840 tttccgagag agcgcttttg
cctacgccat cgcagcagct ggcgtggtgc acgccgtgtc 900 caatgcgtgt
gccctgggca aactgaaggc ctgtggctgt gatgcgtccc ggcgagggga 960
cgaggaggcc ttccgtagga agctgcaccg cttacaactg gatgcactgc agcgtggtaa
1020 gggcctgagc catggggtcc cggaacaccc agccctgccc acagccagcc
caggcctgca 1080 ggactcctgg gagtggggcg gctgcagccc cgacatgggc
ttcggggagc gcttttctaa 1140 ggactttctg gactcccggg agcctcacag
agacatccac gcgagaatga ggcttcacaa 1200 caaccgagtt gggaggcagg
cagtgatgga gaacatgcgg cggaagtgca agtgccacgg 1260 cacgtcaggc
agctgccagc tcaagacgtg ctggcaggtg acgcccgagt tccgcaccgt 1320
gggggcgctg ctgcgcagcc gcttccaccg cgccacgctc atccggccgc acaaccgcaa
1380 cggcggccag ctggagccgg gcccagcggg ggcaccctcg ccggctccgg
gcgctcccgg 1440 gccgcgccga cgggccagcc ccgccgacct ggtctacttc
gaaaagtctc ccgacttctg 1500 cgagcgcgag ccgcgcctgg actcggcggg
caccgtgggc cgcctgtgca acaagagcag 1560 cgccggctcg gatggctgcg
gcagcatgtg ctgcggccgc ggccacaaca tcctgcgcca 1620 gacgcgcagc
gagcgctgcc actgccgctt ccactggtgc tgtttcgtgg tctgcgaaga 1680
gtgccgcatc accgagtggg tcagcgtctg caagtgagcg gcccggggtc ccctgggccc
1740 tgatcgaggt cccctcctgg agcctggccc tctgaggctt acggtcttgg
caaggcagca 1800 tcgccttggc tcttgggaag aggagattgg accacatgat
cttataggaa cccctcagct 1860 ctgaggtctg tgatcgccgg acagtccagg
cctgtctgaa ccccaccact cacttctgtg 1920 ggctctagga ctgactgggt
tcttcctccc tccccgaagc ccagacagtt cagttgggct 1980 gggggttgct
ccacacccta aaacaagcct cagccaggca acccgtcagt ctgtctccat 2040
cctttcaccc cttccctgga gatgggaggt ggggaatgaa tggaagctga cgggcagaga
2100 gaggaggatt aaaaaaaaga aatagacata actgagctga agtaattcca
taaagggccc 2160 agacagcctc ctccaccatt cccttcatca ttcatttaac
aaatatttat tttgcactct 2220 ctttgcggca ctctgggggc ggtggggtgc
gtgggggtgg caatgcaagg cactgaggcc 2280 acagatgtga gtaagcgaga
cacaacactt gtcctcttgg aggttacatt cttgctgggg 2340 ggaggcatgg
gcaataaaca agtaaatata caaac 2375 29 389 PRT Homo sapiens human
Wnt-10b 29 Met Leu Glu Glu Pro Arg Pro Arg Pro Pro Pro Ser Gly Leu
Ala Gly 1 5 10 15 Leu Leu Phe Leu Ala Leu Cys Ser Arg Ala Leu Ser
Asn Glu Ile Leu 20 25 30 Gly Leu Lys Leu Pro Gly Glu Pro Pro Leu
Thr Ala Asn Thr Val Cys 35 40 45 Leu Thr Leu Ser Gly Leu Ser Lys
Arg Gln Leu Gly Leu Cys Leu Arg 50 55 60 Asn Pro Asp Val Thr Ala
Ser Ala Leu Gln Gly Leu His Ile Ala Val 65 70 75 80 His Glu Cys Gln
His Gln Leu Arg Asp Gln Arg Trp Asn Cys Ser Ala 85 90 95 Leu Glu
Gly Gly Gly Arg Leu Pro His His Ser Ala Ile Leu Lys Arg 100 105 110
Gly Phe Arg Glu Ser Ala Phe Ser Phe Ser Met Leu Ala Ala Gly Val 115
120 125 Met His Ala Val Ala Thr Ala Cys Ser Leu Gly Lys Leu Val Ser
Cys 130 135 140 Gly Cys Gly Trp Lys Gly Ser Gly Glu Gln Asp Arg Leu
Arg Ala Lys 145 150 155 160 Leu Leu Gln Leu Gln Ala Leu Ser Arg Gly
Lys Ser Phe Pro His Ser 165 170 175 Leu Pro Ser Pro Gly Pro Gly Ser
Ser Pro Ser Pro Gly Pro Gln Asp 180 185 190 Thr Trp Glu Trp Gly Gly
Cys Asn His Asp Met Asp Phe Gly Glu Lys 195 200 205 Phe Ser Arg Asp
Phe Leu Asp Ser Arg Glu Ala Pro Arg Asp Ile Gln 210 215 220 Ala Arg
Met Arg Ile His Asn Asn Arg Val Gly Arg Gln Val Val Thr 225 230 235
240 Glu Asn Leu Lys Arg Lys Cys Lys Cys His Gly Thr Ser Gly Ser Cys
245 250 255 Gln Phe Lys Thr Cys Trp Arg Ala Ala Pro Glu Phe Arg Ala
Val Gly 260 265 270 Ala Ala Leu Arg Glu Arg Leu Gly Arg Ala Ile Phe
Ile Asp Thr His 275 280 285 Asn Arg Asn Ser Gly Ala Phe Gln Pro Arg
Leu Arg Pro Arg Arg Leu 290 295 300 Ser Gly Glu Leu Val Tyr Phe Glu
Lys Ser Pro Asp Phe Cys Glu Arg 305 310 315 320 Asp Pro Thr Met Gly
Ser Pro Gly Thr Arg Gly Arg Ala Cys Asn Lys 325 330 335 Thr Ser Arg
Leu Leu Asp Gly Cys Gly Ser Leu Cys Cys Gly Arg Gly 340 345 350 His
Asn Val Leu Arg Gln Thr Arg Val Glu Arg Cys His Cys Arg Phe 355 360
365 His Trp Cys Cys Tyr Val Leu Cys Asp Glu Cys Lys Val Thr Glu Trp
370 375 380 Val Asn Val Cys Lys 385 30 2288 DNA Homo sapiens human
Wnt-10b 30 ggggctgcag ctccgtcagc ccggcagagc caccctgagc tcggtgagag
caaagccaga 60 gcccccagtc ctttgctcgc cggcttgcta tctctctcga
tcactccctc ccttcctccc 120 tcccttcctc ccggcggccg cggcggcgct
ggggaagcgg tgaagaggag tggcccggcc 180 ctggaagaat gcggctctga
caaggggaca gaacccagcg cagtctcccc acggtttaag 240 cagcactagt
gaagcccagg caacccaacc gtgcctgtct cggaccccgc acccaaacca 300
ctggaggtcc tgatcgatct gcccaccgga gcctccgggc ttcgacatgc tggaggagcc
360 ccggccgcgg cctccgccct cgggcctcgc gggtctcctg ttcctggcgt
tgtgcagtcg 420 ggctctaagc aatgagattc tgggcctgaa gttgcctggc
gagccgccgc tgacggccaa 480 caccgtgtgc ttgacgctgt ccggcctgag
caagcggcag ctaggcctgt gcctgcgcaa 540 ccccgacgtg acggcgtccg
cgcttcaggg tctgcacatc gcggtccacg agtgtcagca 600 ccagctgcgc
gaccagcgct ggaactgctc cgcgcttgag ggcggcggcc gcctgccgca 660
ccacagcgcc atcctcaagc gcggtttccg agaaagtgct ttttccttct ccatgctggc
720 tgctggggtc atgcacgcag tagccacggc ctgcagcctg ggcaagctgg
tgagctgtgg 780 ctgtggctgg aagggcagtg gtgagcagga tcggctgagg
gccaaactgc tgcagctgca 840 ggcactgtcc cgaggcaaga gtttccccca
ctctctgccc agccctggcc ctggctcaag 900 ccccagccct ggcccccagg
acacatggga atggggtggc tgtaaccatg acatggactt 960 tggagagaag
ttctctcggg atttcttgga ttccagggaa gctccccggg acatccaggc 1020
acgaatgcga atccacaaca acagggtggg gcgccaggtg gtaactgaaa acctgaagcg
1080 gaaatgcaag tgtcatggca catcaggcag ctgccagttc aagacatgct
ggagggcggc 1140 cccagagttc cgggcagtgg gggcggcgtt gagggagcgg
ctgggccggg ccatcttcat 1200 tgatacccac aaccgcaatt ctggagcctt
ccagccccgt ctgcgtcccc gtcgcctctc 1260 aggagagctg gtctactttg
agaagtctcc tgacttctgt gagcgagacc ccactatggg 1320 ctccccaggg
acaaggggcc gggcctgcaa caagaccagc cgcctgttgg atggctgtgg 1380
cagcctgtgc tgtggccgtg ggcacaacgt gctccggcag acacgagttg agcgctgcca
1440 ttgccgcttc cactggtgct gctatgtgct gtgtgatgag tgcaaggtta
cagagtgggt 1500 gaatgtgtgt aagtgagggt cagccttacc ttggggctgg
ggaagaggac tgtgtgagag 1560 gggcgccttt tcagcccttt gctctgattt
ccttccaagg tcactcttgg tccctggaag 1620 cttaaagtat ctacctggaa
acagctttag gggtggtggg ggtcaggtgg actctgggat 1680 gtgtagcctt
ctccccaaca attggagggt cttgagggga agctgccacc cctcttctgc 1740
tccttagaca cctgaatgga ctaagatgaa atgcactgta ttgctcctcc cacttctcaa
1800 ctccagagcc cctttaaccc tgattcatac tccttttggc tggggagtcc
ctatagtttc 1860 accactcctc tcccttgagg gataacccca ggcactgttt
ggagccataa gatctgtatc 1920 tagaaagaga tcacccactc ctatgtacta
tccccaaact cctttactgc agcctgggct 1980 ccctcttgtg ggataatggg
agacagtggt agagaggttt ttcttgggaa agagacagag 2040 tgctgagggg
cactctcccc tgaatcctca gagagttgtc tgtccaggcc cttagggaag 2100
ttgtctcctt ccattcagat gttaatgggg accctccaaa ggaaggggtt ttcccatgac
2160 tcttggagcc tctttttcct tcttcagcag gaagggtggg aagggataat
ttatcatact 2220 gagacttgtt cttggttcct gtttgaaact aaaataaatt
aagttactgg aaaaaaaaaa 2280 aaaaaaaa 2288 31 354 PRT Homo sapiens
human Wnt-11 31 Met Arg Ala Arg Pro Gln Val Cys Glu Ala Leu Leu Phe
Ala Leu Ala 1 5 10 15 Leu Gln Thr Gly Val Cys Tyr Gly Ile Lys Trp
Leu Ala Leu Ser Lys 20 25 30 Thr Pro Ser Ala Leu Ala Leu Asn Gln
Thr Gln His Cys Lys Gln Leu 35 40 45 Glu Gly Leu Val Ser Ala Gln
Val Gln Leu Cys Arg Ser Asn Leu Glu 50 55 60 Leu Met His Thr Val
Val His Ala Ala Arg Glu Val Met Lys Ala Cys 65 70 75 80 Arg Arg Ala
Phe Ala Asp Met Arg Trp Asn Cys Ser Ser Ile Glu Leu 85 90 95 Ala
Pro Asn Tyr Leu Leu Asp Leu Glu Arg Gly Thr Arg Glu Ser Ala 100 105
110 Phe Val Tyr Ala Leu Ser Ala Ala Ala Ile Ser His Ala Ile Ala Arg
115 120 125 Ala Cys Thr Ser Gly Asp Leu Pro Gly Cys Ser Cys Gly Pro
Val Pro 130 135 140 Gly Glu Pro Pro Gly Pro Gly Asn Arg Trp Gly Gly
Cys Ala Asp Asn 145 150 155 160 Leu Ser Tyr Gly Leu Leu Met Gly Ala
Lys Phe Ser Asp Ala Pro Met 165 170 175 Lys Val Lys Lys Thr Gly Ser
Gln Ala Asn Lys Leu Met Arg Leu His 180 185 190 Asn Ser Glu Val Gly
Arg Gln Ala Leu Arg Ala Ser Leu Glu Met Lys 195 200 205 Cys Lys Cys
His Gly Val Ser Gly Ser Cys Ser Ile Arg Thr Cys Trp 210 215 220 Lys
Gly Leu Gln Glu Leu Gln Asp Val Ala Ala Asp Leu Lys Thr Arg 225 230
235 240 Tyr Leu Ser Ala Thr Lys Val Val His Arg Pro Met Gly Thr Arg
Lys 245 250 255 His Leu Val Pro Lys Asp Leu Asp Ile Arg Pro Val Lys
Asp Ser Glu 260 265 270 Leu Val Tyr Leu Gln Ser Ser Pro Asp Phe Cys
Met Lys Asn Glu Lys 275 280 285 Val Gly Ser His Gly Thr Gln Asp Arg
Gln Cys Asn Lys Thr Ser Asn 290 295 300 Gly Ser Asp Ser Cys Asp Leu
Met Cys Cys Gly Arg Gly Tyr Asn Pro 305 310 315 320 Tyr Thr Asp Arg
Val Val Glu Arg Cys His Cys Lys Tyr His Trp Cys 325 330 335 Cys Tyr
Val Thr Cys Arg Arg Cys Glu Arg Thr Val Glu Arg Tyr Val 340 345 350
Cys Lys 32 1927 DNA Homo sapiens human Wnt-11 32 taacccgccg
cctccgctct ccccggctgc aggcggcgtg caggaccagc ggcggccgtg 60
caggcggagg acttcggcgc ggctcctcct gggtgtgacc ccgggcgcgc ccgccgcgcg
120 acgatgaggg cgcggccgca ggtctgcgag gcgctgctct tcgccctggc
gctccagacc 180 ggcgtgtgct atggcatcaa gtggctggcg ctgtccaaga
caccatcggc cctggcactg 240 aaccagacgc aacactgcaa gcagctggag
ggtctggtgt ctgcacaggt gcagctgtgc 300 cgcagcaacc tggagctcat
gcacacggtg gtgcacgccg cccgcgaggt catgaaggcc 360 tgtcgccggg
cctttgccga catgcgctgg aactgctcct ccattgagct cgcccccaac 420
tatttgcttg acctggagag agggacccgg gagtcggcct tcgtgtatgc gctgtcggcc
480 gccgccatca gccacgccat cgcccgggcc tgcacctccg gcgacctgcc
cggctgctcc 540 tgcggccccg tcccaggtga gccacccggg cccgggaacc
gctggggagg atgtgcggac 600 aacctcagct acgggctcct catgggggcc
aagttttccg atgctcctat gaaggtgaaa 660 aaaacaggat cccaagccaa
taaactgatg cgtctacaca acagtgaagt ggggagacag 720 gctctgcgcg
cctctctgga aatgaagtgt aagtgccatg gggtgtctgg ctcctgctcc 780
atccgcacct gctggaaggg gctgcaggag ctgcaggatg tggctgctga cctcaagacc
840 cgatacctgt cggccaccaa ggtagtgcac cgacccatgg gcacccgcaa
gcacctggtg 900 cccaaggacc tggatatccg gcctgtgaag gactcggaac
tcgtctatct gcagagctca 960 cctgacttct gcatgaagaa tgagaaggtg
ggctcccacg ggacacaaga caggcagtgc 1020 aacaagacat ccaacggaag
cgacagctgc gaccttatgt gctgcgggcg tggctacaac 1080 ccctacacag
accgcgtggt cgagcggtgc cactgtaagt accactggtg ctgctacgtc 1140
acctgccgca ggtgtgagcg taccgtggag cgctatgtct gcaagtgagg ccctgccctc
1200 cgccccacgc aggagcgagg actctgctca aggaccctca gcaactgggg
ccaggggcct 1260 ggagacactc catggagctc tgcttgtgaa ttccagatgc
caggcatggg aggcggcttg 1320 tgctttgcct tcacttggaa gccaccagga
acagaaggtc tggccaccct ggaaggaggg 1380 caggacatca aaggaaaccg
acaagattaa aaataacttg gcagcctgag gctctggagt 1440 gcccacaggc
tggtgtaagg agcggggctt gggatcggtg agactgatac agacttgacc 1500
tttcagggcc acagagacca gcctccggga aggggtctgc ccgccttctt cagaatgttc
1560 tgcgggaccc cctggcccac cctggggtct gagcctgctg ggcccaccac
atggaatcac 1620 tagcttgggt tgtaaatgtt ttcttttgtt ttttgctttt
tcttcctttg ggatgtggaa 1680 gctacagaaa tatttataaa acatagcttt
ttctttgggg tggcacttct caattcctct 1740 ttatatattt tatatatata
aatatatatg tatatatata atgatctcta ttttaaaact 1800 agctttttaa
gcagctgtat gaaataaatg ctgagtgagc cccagcccgc ccctgcagtt 1860
cccggcctcg tcaagtgaac tcggcagacc ctggggctgg cagagggagc tctccagttt
1920 ccaggca 1927 33 365 PRT Homo sapiens human Wnt-14 33 Met Leu
Asp Gly Ser Pro Leu Ala Arg Trp Leu Ala Ala Ala Phe Gly 1 5 10 15
Leu Thr Leu Leu Leu Ala Ala Leu Arg Pro Ser Ala Ala Tyr Phe Gly 20
25 30 Leu Thr Gly Ser Glu Pro Leu Thr Ile Leu Pro Leu Thr Leu Glu
Pro 35 40 45 Glu Ala Ala Ala Gln Ala His Tyr Lys Ala Cys Asp Arg
Leu Lys Leu 50 55 60 Glu Arg Lys Gln Arg Arg Met Cys Arg Arg Asp
Pro Gly Val Ala Glu 65 70 75 80 Thr Leu Val Glu Ala Val Ser Met Ser
Ala Leu Glu Cys Gln Phe Gln 85 90 95 Phe Arg Phe Glu Arg Trp Asn
Cys Thr Leu Glu Gly Arg Tyr Arg Ala 100 105 110 Ser Leu Leu Lys Arg
Gly Phe Lys Glu Thr Ala Phe Leu Tyr Ala Ile 115 120 125 Ser Ser Ala
Gly Leu Thr His Ala Leu Ala Lys Ala Cys Ser Ala Gly 130 135 140 Arg
Met Glu Arg Cys Thr Cys Asp Glu Ala Pro Asp Leu Glu Asn Arg 145 150
155 160 Glu Ala Trp Gln Trp Gly Gly Cys Gly Asp Asn Leu Lys Tyr Ser
Ser 165 170 175 Lys Phe Val Lys Glu Phe Leu Gly Arg Arg Ser Ser Lys
Asp Leu Arg 180 185 190 Ala Arg Val Asp Phe His Asn Asn Leu Val Gly
Val Lys Val Ile Lys 195 200 205 Ala Gly Val Glu Thr Thr Cys Lys Cys
His Gly Val Ser Gly Ser Cys 210 215 220 Thr Val Arg Thr Cys Trp Arg
Gln Leu Ala Pro Phe His Glu Val Gly 225 230 235 240 Lys His Leu Lys
His Lys Tyr Glu Thr Ala Leu Lys Val Gly Ser Thr 245 250 255 Thr Asn
Glu Ala Ala Gly Glu Ala Gly Ala Ile Ser Pro Pro Arg Gly 260 265 270
Arg Ala Ser Gly Ala Gly Gly Ser Asp Pro Leu Pro Arg Thr Pro Glu 275
280 285 Leu Val His Leu Asp Asp Ser Pro Ser Phe Cys Leu Ala Gly Arg
Phe 290 295 300 Ser Pro Gly Thr Ala Gly Arg Arg Cys His Arg Glu Lys
Asn Cys Glu 305 310 315 320 Ser Ile Cys Cys Gly Arg Gly His Asn Thr
Gln Ser Arg Val Val Thr 325 330 335 Arg Pro Cys Gln Cys Gln Val Arg
Trp Cys Cys Tyr Val Glu Cys Arg 340 345 350 Gln Cys Thr Gln Arg Glu
Glu Val Tyr Thr Cys Lys Gly 355 360 365 34 1631 DNA Homo sapiens
human Wnt-14 34 ggcgcggcaa gatgctggat gggtccccgc tggcgcgctg
gctggccgcg gccttcgggc 60 tgacgctgct gctcgccgcg ctgcgccctt
cggccgccta cttcgggctg acgggcagcg 120 agcccctgac catcctcccg
ctgaccctgg agccagaggc ggccgcccag gcgcactaca 180 aggcctgcga
ccggctgaag ctggagcgga agcagcggcg catgtgccgc cgggacccgg 240
gcgtggcaga gacgctggtg gaggccgtga gcatgagtgc gctcgagtgc cagttccagt
300 tccgctttga gcgctggaac tgcacgctgg agggccgcta ccgggccagc
ctgctcaagc 360 gaggcttcaa ggagactgcc ttcctctatg ccatctcctc
ggctggcctg acgcacgcac 420 tggccaaggc gtgcagcgcg ggccgcatgg
agcgctgtac ctgcgatgag gcacccgacc 480 tggagaaccg tgaggcctgg
cagtgggggg gctgcggaga caaccttaag tacagcagca 540 agttcgtcaa
ggaattcctg ggcagacggt caagcaagga tctgcgagcc cgtgtggact 600
tccacaacaa cctcgtgggt gtgaaggtga tcaaggctgg ggtggagacc acctgcaagt
660 gccacggcgt gtcaggctca tgcacggtgc ggacctgctg gcggcagttg
gcgcctttcc 720 atgaggtggg caagcatctg aagcacaagt atgagacggc
actcaaggtg ggcagcacca 780 ccaatgaagc tgccggcgag gcaggtgcca
tctccccacc acggggccgt gcctcggggg 840 caggtggcag cgacccgctg
ccccgcactc cagagctggt gcacctggat gactcgccta 900 gcttctgcct
ggctggccgc ttctccccgg gcaccgctgg ccgtaggtgc caccgtgaga 960
agaactgcga gagcatctgc tgtggccgcg gccataacac acagagccgg gtggtgacaa
1020 ggccctgcca
gtgccaggtg cgttggtgct gctatgtgga gtgcaggcag tgcacgcagc 1080
gtgaggaggt ctacacctgc aagggctgag ttcccaggcc ctgccagccc tgctgcacag
1140 ggtgcaggca ttgcacacgg tgtgaagggt ctacacctgc acaggctgag
ttcctgggct 1200 cgaccagccc agctgcgtgg ggtacaggca ttgcacacag
tgtgaatggg tctacacctg 1260 catgggctga gtccctgggc tcagacctag
cagcgtgggg tagtccctgg gctcagtcct 1320 agctgcatgg ggtgcaggca
ttgcacagag catgaatggg cctacacctg ccaaggctga 1380 atccctgggc
ccagccagcc ctgctgcaca tggcacaggc attgcacacg gtgtgaggag 1440
tgtacacctg caagggctga ggccctgggc ccagtcagcc ctgctgctca gagtgcaggc
1500 attgcacatg gtgtgagaag gtctacacct gcaagggacg agtccccggg
cctggccaac 1560 cctgctgtgc agggtgaggg ccatgcatgc tagtatgagg
ggtctacacc tgcaaggact 1620 gagaggcttt t 1631 35 357 PRT Homo
sapiens human Wnt-15 35 Met Arg Pro Pro Pro Ala Leu Ala Leu Ala Gly
Leu Cys Leu Leu Ala 1 5 10 15 Leu Pro Ala Ala Ala Ala Ser Tyr Phe
Gly Leu Thr Gly Arg Glu Val 20 25 30 Leu Thr Pro Phe Pro Gly Leu
Gly Thr Ala Ala Ala Pro Ala Gln Gly 35 40 45 Gly Ala His Leu Lys
Gln Cys Asp Leu Leu Lys Leu Ser Arg Arg Gln 50 55 60 Lys Gln Leu
Cys Arg Arg Glu Pro Gly Leu Ala Glu Thr Leu Arg Asp 65 70 75 80 Ala
Ala His Leu Gly Leu Leu Glu Cys Gln Phe Gln Phe Arg His Glu 85 90
95 Arg Trp Asn Cys Ser Leu Glu Gly Arg Met Gly Leu Leu Lys Arg Gly
100 105 110 Phe Lys Glu Thr Ala Phe Leu Tyr Ala Val Ser Ser Ala Ala
Leu Thr 115 120 125 His Thr Leu Ala Arg Ala Cys Ser Ala Gly Arg Met
Glu Arg Cys Thr 130 135 140 Cys Asp Asp Ser Pro Gly Leu Glu Ser Arg
Gln Ala Trp Gln Trp Gly 145 150 155 160 Val Cys Gly Asp Asn Leu Lys
Tyr Ser Thr Lys Phe Leu Ser Asn Phe 165 170 175 Leu Gly Ser Lys Arg
Gly Asn Lys Asp Leu Arg Ala Arg Ala Asp Ala 180 185 190 His Asn Thr
His Val Gly Ile Lys Ala Val Lys Ser Gly Leu Arg Thr 195 200 205 Thr
Cys Lys Cys His Gly Val Ser Gly Ser Cys Ala Val Arg Thr Cys 210 215
220 Trp Lys Gln Leu Ser Pro Phe Arg Glu Thr Gly Gln Val Leu Lys Leu
225 230 235 240 Arg Tyr Asp Ser Ala Val Lys Val Ser Ser Ala Thr Asn
Glu Ala Leu 245 250 255 Gly Arg Leu Glu Leu Trp Ala Pro Ala Arg Gln
Gly Ser Leu Thr Lys 260 265 270 Gly Leu Ala Pro Arg Ser Gly Asp Leu
Val Tyr Met Glu Asp Ser Pro 275 280 285 Ser Phe Cys Arg Pro Ser Lys
Tyr Ser Pro Gly Thr Ala Gly Arg Val 290 295 300 Cys Ser Arg Glu Ala
Ser Cys Ser Ser Leu Cys Cys Gly Arg Gly Tyr 305 310 315 320 Asp Thr
Gln Ser Arg Leu Val Ala Phe Ser Cys His Cys Gln Val Gln 325 330 335
Trp Cys Cys Tyr Val Glu Cys Gln Gln Cys Val Gln Glu Glu Leu Val 340
345 350 Tyr Thr Cys Lys His 355 36 1464 DNA Homo sapiens human
Wnt-15 36 gcgaggagat gctagagggc gcagcgccgc cagcaccatg cgccccccgc
ccgcgctggc 60 cctggccggg ctctgcctgc tggcgctgcc cgccgccgcc
gcctcctact tcggcctgac 120 cgggcgggaa gtcctgacgc ccttcccagg
attgggcact gcggcagccc cggcacaggg 180 cggggcccac ctgaagcagt
gtgacctgct gaagctgtcc cggcggcaga agcagctctg 240 ccggagggag
cccggcctgg ctgagaccct gagggatgct gcgcacctcg gcctgcttga 300
gtgccagttt cagttccggc atgagcgctg gaactgtagc ctggagggca ggatgggcct
360 gctcaagaga ggcttcaaag agacagcttt cctgtacgcg gtgtcctctg
ccgccctcac 420 ccacaccctg gcccgggcct gcagcgctgg gcgcatggag
cgctgcacct gtgatgactc 480 tccggggctg gagagccggc aggcctggca
gtggggcgtg tgcggtgaca acctcaagta 540 cagcaccaag tttctgagca
acttcctggg gtccaagaga ggaaacaagg acctgcgggc 600 acgggcagac
gcccacaata cccacgtggg catcaaggct gtgaagagtg gcctcaggac 660
cacgtgtaag tgccatggcg tatcaggctc ctgtgccgtg cgcacctgct ggaagcagct
720 ctccccgttc cgtgagacgg gccaggtgct gaaactgcgc tatgactcgg
ctgtcaaggt 780 gtccagtgcc accaatgagg ccttgggccg cctagagctg
tgggcccctg ccaggcaggg 840 cagcctcacc aaaggcctgg ccccaaggtc
tggggacctg gtgtacatgg aggactcacc 900 cagcttctgc cggcccagca
agtactcacc tggcacagca ggtagggtgt gctcccggga 960 ggccagctgc
agcagcctgt gctgcgggcg gggctatgac acccagagcc gcctggtggc 1020
cttctcctgc cactgccagg tgcagtggtg ctgctacgtg gagtgccagc aatgtgtgca
1080 ggaggagctt gtgtacacct gcaagcacta ggcctactgc ccagcaagcc
agtctggcac 1140 tgccaggacc tcctgtggca cccttcaagc tgcccagccg
gccctctggg cagactgtca 1200 tcacatgcat gcataaaccg gcatgtgtgc
caatgcacac gagtgtgcca ctcaccacca 1260 ttccttggcc agccttttgc
ctccctcgat actcaacaaa gagaagcaaa gcctcctccc 1320 ttaacccaag
catccccaac cttgttgagg acttggagag gagggcagag tgagaaagac 1380
atggagggaa ataagggaga ccaagagcac agcaggactg aaattttgga cgggagagag
1440 gggctattcc atcttgcttc ctgg 1464 37 365 PRT Homo sapiens human
Wnt-16 37 Met Asp Arg Ala Ala Leu Leu Gly Leu Ala Arg Leu Cys Ala
Leu Trp 1 5 10 15 Ala Ala Leu Leu Val Leu Phe Pro Tyr Gly Ala Gln
Gly Asn Trp Met 20 25 30 Trp Leu Gly Ile Ala Ser Phe Gly Val Pro
Glu Lys Leu Gly Cys Ala 35 40 45 Asn Leu Pro Leu Asn Ser Arg Gln
Lys Glu Leu Cys Lys Arg Lys Pro 50 55 60 Tyr Leu Leu Pro Ser Ile
Arg Glu Gly Ala Arg Leu Gly Ile Gln Glu 65 70 75 80 Cys Gly Ser Gln
Phe Arg His Glu Arg Trp Asn Cys Met Ile Thr Ala 85 90 95 Ala Ala
Thr Thr Ala Pro Met Gly Ala Ser Pro Leu Phe Gly Tyr Glu 100 105 110
Leu Ser Ser Gly Thr Lys Glu Thr Ala Phe Ile Tyr Ala Val Met Ala 115
120 125 Ala Gly Leu Val His Ser Val Thr Arg Ser Cys Ser Ala Gly Asn
Met 130 135 140 Thr Glu Cys Ser Cys Asp Thr Thr Leu Gln Asn Gly Gly
Ser Ala Ser 145 150 155 160 Glu Gly Trp His Trp Gly Gly Cys Ser Asp
Asp Val Gln Tyr Gly Met 165 170 175 Trp Phe Ser Arg Lys Phe Leu Asp
Phe Pro Ile Gly Asn Thr Thr Gly 180 185 190 Lys Glu Asn Lys Val Leu
Leu Ala Met Asn Leu His Asn Asn Glu Ala 195 200 205 Gly Arg Gln Ala
Val Ala Lys Leu Met Ser Val Asp Cys Arg Cys His 210 215 220 Gly Val
Ser Gly Ser Cys Ala Val Lys Thr Cys Trp Lys Thr Met Ser 225 230 235
240 Ser Phe Glu Lys Ile Gly His Leu Leu Lys Asp Lys Tyr Glu Asn Ser
245 250 255 Ile Gln Ile Ser Asp Lys Thr Lys Arg Lys Met Arg Arg Arg
Glu Lys 260 265 270 Asp Gln Arg Lys Ile Pro Ile His Lys Asp Asp Leu
Leu Tyr Val Asn 275 280 285 Lys Ser Pro Asn Tyr Cys Val Glu Asp Lys
Lys Leu Gly Ile Pro Gly 290 295 300 Thr Gln Gly Arg Glu Cys Asn Arg
Thr Ser Glu Gly Ala Asp Gly Cys 305 310 315 320 Asn Leu Leu Cys Cys
Gly Arg Gly Tyr Asn Thr His Val Val Arg His 325 330 335 Val Glu Arg
Cys Glu Cys Lys Phe Ile Trp Cys Cys Tyr Val Arg Cys 340 345 350 Arg
Arg Cys Glu Ser Met Thr Asp Val His Thr Cys Lys 355 360 365 38 3132
DNA Homo sapiens human Wnt-16 38 cccgcatctc ctgcacatct ccacccctgc
gcaggaggag atccccaggc tgctctctcc 60 atctctccta cagctccctg
caaacgaggg ggaagctgct gagagtccct atcactgctg 120 gccttttaat
gttgtatgca aggaggaaga gggcgaggga taacttggtg ctggacaact 180
gacctgcggc ccgaagggcc tctggggagg gggtgcaaaa gaggagcggc tgggctgggg
240 gactccatgc gggggcgatg gacagggcgg cgctcctggg actggcccgc
ttgtgcgcgc 300 tgtgggcagc cctgctcgtg ctgttcccct acggagccca
aggaaactgg atgtggttgg 360 gcattgcctc cttcggggtt ccagagaagc
tgggctgcgc caatttgccg ctgaacagcc 420 gccagaagga gctgtgcaag
aggaaaccgt acctgctgcc gagcatccga gagggcgccc 480 ggctgggcat
tcaggagtgc gggagccagt tcagacacga gagatggaac tgcatgatca 540
ccgccgccgc cactaccgcc ccgatgggcg ccagccccct ctttggctac gagctgagca
600 gcggcaccaa agagacagca tttatttatg ctgtgatggc tgcaggcctg
gtgcattctg 660 tgaccaggtc atgcagtgca ggcaacatga cagagtgttc
ctgtgacacc accttgcaga 720 acggcggctc agcaagtgaa ggctggcact
gggggggctg ctccgatgat gtccagtatg 780 gcatgtggtt cagcagaaag
ttcctagatt tccccatcgg aaacaccacg ggcaaagaaa 840 acaaagtact
attagcaatg aacctacata acaatgaagc tggaaggcag gctgtcgcca 900
agttgatgtc agtagactgc cgctgccacg gagtttccgg ctcctgtgct gtgaaaacat
960 gctggaaaac catgtcttct tttgaaaaga ttggccattt gttgaaggat
aaatatgaaa 1020 acagtatcca gatatcagac aaaacaaaga ggaaaatgcg
caggagagaa aaagatcaga 1080 ggaaaatacc aatccataag gatgatctgc
tctatgttaa taagtctccc aactactgtg 1140 tagaagataa gaaactggga
atcccaggga cacaaggcag agaatgcaac cgtacatcag 1200 agggtgcaga
tggctgcaac ctcctctgct gtggccgagg ttacaacacc catgtggtca 1260
ggcacgtgga gaggtgtgag tgtaagttca tctggtgctg ctatgtccgt tgcaggaggt
1320 gtgaaagcat gactgatgtc cacacttgca agtaaccact ccatccagcc
ttgggcaaga 1380 tgcctcagca atatacaatg gcattgcaac cagagaggtg
cccatccctg tgcagcgcta 1440 gtaaagttga ctcttgcagt ggaatcccta
gaaccttgga cctgagagtt tcccttacct 1500 gatcgacata ttttccttta
tctgatcaac ccatcaatca tgtggatttc ttgggattct 1560 aatgttgaaa
aggtttatat tcaccttttg atgatttggg gaatatatat tgacatacaa 1620
ggaagataat ctgtttccta agcaagaaat aacaggaaag atcccttatg ccaggaggcc
1680 tgccatactc aggataagat ccttgaatat ggaacttagt tacaggactc
aataatggtg 1740 ggtgaacatt agtcattttt aaaagacacc tcttatagca
ataaggagac attaacatga 1800 atctcattta ttctctcagt attttaactg
aagaaattat actgtttgtg tgtggataga 1860 agatgttgaa aagttaacat
aagcattggg tgctgactta ccctttcatg tacttccaaa 1920 gaaaggtaat
caaaaagaat cttcttaagt gatataatat ccctaaaaaa atgatcatta 1980
cagatgttta gtgacaaaga atcaatatgt aaaaagtata atgaatgatt tagattttaa
2040 gtgccttttc actgggagaa tctggaaaaa cctccataag gtatatagca
atctttgatc 2100 tttagattca tacttttatc acagatcagt ttcaactgtt
aaaaacccac ctctgagata 2160 ctggggggag gatcctgaaa catgcgggaa
aaggagaggt aaacagtgga ggtaaaaata 2220 taatttcata cattgtaaag
aaaagcaccc tttaaatgtg taaagacagt gttttgtaaa 2280 gaattttgtt
taaaaagttt ctattttgta aatacagtac ttaagttata tgatttatat 2340
taaaacattt attgacaaag cctaagagct aaggcagtaa aattatctca taaataatat
2400 tagcttattt tttttcatac tattaatgct atttttttgg acatcgaaga
gaatttaact 2460 tagcagttag ttatatggat gtgtatttct tgctaaaatg
acagttttat atgttataga 2520 ttaaaatatg ttgcaaaata tcaaaaattt
gtgttatttc agcagtaaga ttaattgaat 2580 tctcttttca cattagttat
gcttaactca taaggttatt ataataaatt atattagtaa 2640 aagtcttaac
tggaaaaaag aatctaaatc agaatagtga tcaatttgtg gatttgatat 2700
cctggatatt tattatattt tatgtaatgc tgcatttcta tttgaatgtt aagtggtctt
2760 tcttgttttt aatattcatg catgtatatt catcatattt tacaaggttc
ctggtaaaaa 2820 ttacagggct ctatttaagg atgtatttta atgtaaatgc
ttatgttttt tatgaattgt 2880 taaatatttc agtattatat agaaaaaaat
agatttttaa aattcagaat ggacaaagag 2940 aatattcatt ttcttattaa
taagataaag aaatgtttcc ctgccccaca gtcttcattc 3000 tatttctctt
taattttatt cactgaggca gagaaacaat ttttgaaaaa gagcaaaccc 3060
atggaaaatg tctcagatct aatattaaaa tcaagactaa gcatttaact gtgaaaaaaa
3120 aaaaaaaaaa aa 3132 39 647 PRT Homo sapiens human frizzled1
(Fzd1) 39 Met Ala Glu Glu Glu Ala Pro Lys Lys Ser Arg Ala Ala Gly
Gly Gly 1 5 10 15 Ala Ser Trp Glu Leu Cys Ala Gly Ala Leu Ser Ala
Arg Leu Ala Glu 20 25 30 Glu Gly Ser Gly Asp Ala Gly Gly Arg Arg
Arg Pro Pro Val Asp Pro 35 40 45 Arg Arg Leu Ala Arg Gln Leu Leu
Leu Leu Leu Trp Leu Leu Glu Ala 50 55 60 Pro Leu Leu Leu Gly Val
Arg Ala Gln Ala Ala Gly Gln Gly Pro Gly 65 70 75 80 Gln Gly Pro Gly
Pro Gly Gln Gln Pro Pro Pro Pro Pro Gln Gln Gln 85 90 95 Gln Ser
Gly Gln Gln Tyr Asn Gly Glu Arg Gly Ile Ser Val Pro Asp 100 105 110
His Gly Tyr Cys Gln Pro Ile Ser Ile Pro Leu Cys Thr Asp Ile Ala 115
120 125 Tyr Asn Gln Thr Ile Met Pro Asn Leu Leu Gly His Thr Asn Gln
Glu 130 135 140 Asp Ala Gly Leu Glu Val His Gln Phe Tyr Pro Leu Val
Lys Val Gln 145 150 155 160 Cys Ser Ala Glu Leu Lys Phe Phe Leu Cys
Ser Met Tyr Ala Pro Val 165 170 175 Cys Thr Val Leu Glu Gln Ala Leu
Pro Pro Cys Arg Ser Leu Cys Glu 180 185 190 Arg Ala Arg Gln Gly Cys
Glu Ala Leu Met Asn Lys Phe Gly Phe Gln 195 200 205 Trp Pro Asp Thr
Leu Lys Cys Glu Lys Phe Pro Val His Gly Ala Gly 210 215 220 Glu Leu
Cys Val Gly Gln Asn Thr Ser Asp Lys Gly Thr Pro Thr Pro 225 230 235
240 Ser Leu Leu Pro Glu Phe Trp Thr Ser Asn Pro Gln His Gly Gly Gly
245 250 255 Gly His Arg Gly Gly Phe Pro Gly Gly Ala Gly Ala Ser Glu
Arg Gly 260 265 270 Lys Phe Ser Cys Pro Arg Ala Leu Lys Val Pro Ser
Tyr Leu Asn Tyr 275 280 285 His Phe Leu Gly Glu Lys Asp Cys Gly Ala
Pro Cys Glu Pro Thr Lys 290 295 300 Val Tyr Gly Leu Met Tyr Phe Gly
Pro Glu Glu Leu Arg Phe Ser Arg 305 310 315 320 Thr Trp Ile Gly Ile
Trp Ser Val Leu Cys Cys Ala Ser Thr Leu Phe 325 330 335 Thr Val Leu
Thr Tyr Leu Val Asp Met Arg Arg Phe Ser Tyr Pro Glu 340 345 350 Arg
Pro Ile Ile Phe Leu Ser Gly Cys Tyr Thr Ala Val Ala Val Ala 355 360
365 Tyr Ile Ala Gly Phe Leu Leu Glu Asp Arg Val Val Cys Asn Asp Lys
370 375 380 Phe Ala Glu Asp Gly Ala Arg Thr Val Ala Gln Gly Thr Lys
Lys Glu 385 390 395 400 Gly Cys Thr Ile Leu Phe Met Met Leu Tyr Phe
Phe Ser Met Ala Ser 405 410 415 Ser Ile Trp Trp Val Ile Leu Ser Leu
Thr Trp Phe Leu Ala Ala Gly 420 425 430 Met Lys Trp Gly His Glu Ala
Ile Glu Ala Asn Ser Gln Tyr Phe His 435 440 445 Leu Ala Ala Trp Ala
Val Pro Ala Ile Lys Thr Ile Thr Ile Leu Ala 450 455 460 Leu Gly Gln
Val Asp Gly Asp Val Leu Ser Gly Val Cys Phe Val Gly 465 470 475 480
Leu Asn Asn Val Asp Ala Leu Arg Gly Phe Val Leu Ala Pro Leu Phe 485
490 495 Val Tyr Leu Phe Ile Gly Thr Ser Phe Leu Leu Ala Gly Phe Val
Ser 500 505 510 Leu Phe Arg Ile Arg Thr Ile Met Lys His Asp Gly Thr
Lys Thr Glu 515 520 525 Lys Leu Glu Lys Leu Met Val Arg Ile Gly Val
Phe Ser Val Leu Tyr 530 535 540 Thr Val Pro Ala Thr Ile Val Ile Ala
Cys Tyr Phe Tyr Glu Gln Ala 545 550 555 560 Phe Arg Asp Gln Trp Glu
Arg Ser Trp Val Ala Gln Ser Cys Lys Ser 565 570 575 Tyr Ala Ile Pro
Cys Pro His Leu Gln Ala Gly Gly Gly Ala Pro Pro 580 585 590 His Pro
Pro Met Ser Pro Asp Phe Thr Val Phe Met Ile Lys Tyr Leu 595 600 605
Met Thr Leu Ile Val Gly Ile Thr Ser Gly Phe Trp Ile Trp Ser Gly 610
615 620 Lys Thr Leu Asn Ser Trp Arg Lys Phe Tyr Thr Arg Leu Thr Asn
Ser 625 630 635 640 Lys Gln Gly Glu Thr Thr Val 645 40 4350 DNA
Homo sapiens human frizzled1 (Fzd1) 40 agttgaggga ttgacacaaa
tggtcaggcg gcggcggcgg agaaggaggc ggaggcgcag 60 gggggagccg
agcccgctgg gctgcggaga gttgcgctct ctacggggcc gcggccacta 120
gcgcggcgcc gccagccggg agccagcgag ccgagggcca ggaaggcggg acacgacccc
180 ggcgcgccct agccacccgg gttctccccg ccgcccgcgc ttcatgaatc
gcaagtttcc 240 gcggcggcgg cggctgcggt acgcagaaca ggagccgggg
gagcgggccg aaagcggctt 300 gggctcgacg gagggcaccc gcgcagaggt
ctccctggcc gcagggggag ccgccgccgg 360 ccgtgcccct ggcagcccca
gcggagcggc gccaagagag gagccgagaa agtatggctg 420 aggaggaggc
gcctaagaag tcccgggccg ccggcggtgg cgcgagctgg gaactttgtg 480
ccggggcgct ctcggcccgg ctggcggagg agggcagcgg ggacgccggt ggccgccgcc
540 gcccgccagt tgacccccgg cgattggcgc gccagctgct gctgctgctt
tggctgctgg 600 aggctccgct gctgctgggg gtccgggccc aggcggcggg
ccaggggcca ggccaggggc 660 ccgggccggg gcagcaaccg ccgccgccgc
ctcagcagca acagagcggg cagcagtaca 720 acggcgagcg gggcatctcc
gtcccggacc acggctattg ccagcccatc tccatcccgc 780 tgtgcacgga
catcgcgtac aaccagacca tcatgcccaa cctgctgggc cacacgaacc 840
aggaggacgc gggcctggag gtgcaccagt tctaccctct agtgaaagtg cagtgttccg
900 ctgagctcaa gttcttcctg tgctccatgt acgcgcccgt gtgcaccgtg
ctagagcagg 960 cgctgccgcc ctgccgctcc ctgtgcgagc gcgcgcgcca
gggctgcgag gcgctcatga 1020 acaagttcgg cttccagtgg ccagacacgc
tcaagtgtga gaagttcccg gtgcacggcg 1080
ccggcgagct gtgcgtgggc cagaacacgt ccgacaaggg caccccgacg ccctcgctgc
1140 ttccagagtt ctggaccagc aaccctcagc acggcggcgg agggcaccgt
ggcggcttcc 1200 cggggggcgc cggcgcgtcg gagcgaggca agttctcctg
cccgcgcgcc ctcaaggtgc 1260 cctcctacct caactaccac ttcctggggg
agaaggactg cggcgcacct tgtgagccga 1320 ccaaggtgta tgggctcatg
tacttcgggc ccgaggagct gcgcttctcg cgcacctgga 1380 ttggcatttg
gtcagtgctg tgctgcgcct ccacgctctt cacggtgctt acgtacctgg 1440
tggacatgcg gcgcttcagc tacccggagc ggcccatcat cttcttgtcc ggctgttaca
1500 cggccgtggc cgtggcctac atcgccggct tcctcctgga agaccgagtg
gtgtgtaatg 1560 acaagttcgc cgaggacggg gcacgcactg tggcgcaggg
caccaagaag gagggctgca 1620 ccatcctctt catgatgctc tacttcttca
gcatggccag ctccatctgg tgggtgatcc 1680 tgtcgctcac ctggttcctg
gcggctggca tgaagtgggg ccacgaggcc atcgaagcca 1740 actcacagta
ttttcacctg gccgcctggg ctgtgccggc catcaagacc atcaccatcc 1800
tggcgctggg ccaggtggac ggcgatgtgc tgagcggagt gtgcttcgtg gggcttaaca
1860 acgtggacgc gctgcgtggc ttcgtgctgg cgcccctctt cgtgtacctg
tttatcggca 1920 cgtcctttct gctggccggc tttgtgtcgc tcttccgcat
ccgcaccatc atgaagcacg 1980 atggcaccaa gaccgagaag ctggagaagc
tcatggtgcg cattggcgtc ttcagcgtgc 2040 tgtacactgt gccagccacc
atcgtcatcg cctgctactt ctacgagcag gccttccggg 2100 accagtggga
acgcagctgg gtggcccaga gctgcaagag ctacgctatc ccctgccctc 2160
acctccaggc gggcggaggc gccccgccgc acccgcccat gagcccggac ttcacggtct
2220 tcatgattaa gtaccttatg acgctgatcg tgggcatcac gtcgggcttc
tggatctggt 2280 ccggcaagac cctcaactcc tggaggaagt tctacacgag
gctcaccaac agcaaacaag 2340 gggagactac agtctgagac ccggggctca
gcccatgccc aggcctcggc cggggcgcag 2400 cgatccccca aagccagcgc
cgtggagttc gtgccaatcc tgacatctcg aggtttcctc 2460 actagacaac
tctctttcgc aggctccttt gaacaactca gctcctgcaa aagcttccgt 2520
ccctgaggca aaaggacacg agggcccgac tgccagaggg aggatggaca gacctcttgc
2580 cctcacactc tggtaccagg actgttcgct tttatgattg taaatagcct
gtgtaagatt 2640 tttgtaagta tatttgtatt taaatgacga ccgatcacgc
gtttttcttt ttcaaaagtt 2700 tttaattatt tagggcggtt taaccatttg
aggcttttcc ttcttgccct tttcggagta 2760 ttgcaaagga gctaaaactg
gtgtgcaacc gcacagcgct cctggtcgtc ctcgcgcgcc 2820 tctccctacc
acgggtgctc gggacggctg ggcgccagct ccggggcgag ttcagcactg 2880
cggggtgcga ctagggctgc gctgccaggg tcacttcccg cctcctcctt ttgccccctc
2940 cccctccttc tgtcccctcc ctttctttcc tggcttgagg taggggctct
taaggtacag 3000 aactccacaa accttccaaa tctggaggag ggcccccata
cattacaatt cctcccttgc 3060 tcggcggtgg attgcgaagg cccgtccctt
cgacttcctg aagctggatt tttaactgtc 3120 cagaactttc ctccaacttc
atgggggccc acgggtgtgg gcgctggcag tctcagcctc 3180 cctccacggt
caccttcaac gcccagacac tcccttctcc caccttagtt ggttacaggg 3240
tgagtgagat aaccaatgcc aaactttttg aagtctaatt tttgaggggt gagctcattt
3300 cattctctag tgtctaaaac ctggtatggg tttggccagc gtcatggaaa
gatgtggtta 3360 ctgagatttg ggaagaagca tgaagctttg tgtgggttgg
aagagactga agatatgggt 3420 tataaaatgt taattctaat tgcatacgga
tgcctggcaa ccttgccttt gagaatgaga 3480 cagcctgcgc ttagatttta
ccggtctgta aaatggaaat gttgaggtca cctggaaagc 3540 tttgttaagg
agttgatgtt tgctttcctt aacaagacag caaaacgtaa acagaaattg 3600
aaaacttgaa ggatatttca gtgtcatgga cttcctcaaa atgaagtgct attttcttat
3660 ttttaatcaa ataactagac atatatcaga aactttaaaa tgtaaaagtt
gtacactttc 3720 aacattttat tacgattatt attcagcagc acattctgag
gggggaacaa ttcacaccac 3780 caataataac ctggtaagat ttcaggaggt
aaagaaggtg gaataattga cggggagata 3840 gcgcctgaaa taaacaaaat
atgggcatgc atgctaaagg gaaaatgtgt gcaggtctac 3900 tgcattaaat
cctgtgtgct cctcttttgg atttacagaa atgtgtcaaa tgtaaatctt 3960
tcaaagccat ttaaaaatat tcactttagt tctctgtgaa gaagaggaga aaagcaatcc
4020 tcctgattgt attgttttaa actttaagaa tttatcaaaa tgccggtact
taggacctaa 4080 atttatctat gtctgtcata cgctaaaatg atattggtct
ttgaatttgg tatacattta 4140 ttctgttcac tatcacaaaa tcatctatat
ttatagagga atagaagttt atatatatat 4200 aataccatat ttttaatttc
acaaataaaa aattcaaagt tttgtacaaa attatatgga 4260 ttttgtgcct
gaaaataata gagcttgagc tgtctgaact attttacatt ttatggtgtc 4320
tcatagccaa tcccacagtg taaaaattca 4350 41 565 PRT Homo sapiens human
frizzled2 (Fzd2) 41 Met Arg Pro Arg Ser Ala Leu Pro Arg Leu Leu Leu
Pro Leu Leu Leu 1 5 10 15 Leu Pro Ala Ala Gly Pro Ala Gln Phe His
Gly Glu Lys Gly Ile Ser 20 25 30 Ile Pro Asp His Gly Phe Cys Gln
Pro Ile Ser Ile Pro Leu Cys Thr 35 40 45 Asp Ile Ala Tyr Asn Gln
Thr Ile Met Pro Asn Leu Leu Gly His Thr 50 55 60 Asn Gln Glu Asp
Ala Gly Leu Glu Val His Gln Phe Tyr Pro Leu Val 65 70 75 80 Lys Val
Gln Cys Ser Pro Glu Leu Arg Phe Phe Leu Cys Ser Met Tyr 85 90 95
Ala Pro Val Cys Thr Val Leu Glu Gln Ala Ile Pro Pro Cys Arg Ser 100
105 110 Ile Cys Glu Arg Ala Arg Gln Gly Cys Glu Ala Leu Met Asn Lys
Phe 115 120 125 Gly Phe Gln Trp Pro Glu Arg Leu Arg Cys Glu His Phe
Pro Arg His 130 135 140 Gly Ala Glu Gln Ile Cys Val Gly Gln Asn His
Ser Glu Asp Gly Ala 145 150 155 160 Pro Ala Leu Leu Thr Thr Ala Pro
Pro Pro Gly Leu Gln Pro Gly Ala 165 170 175 Gly Gly Thr Pro Gly Gly
Pro Gly Gly Gly Gly Ala Pro Pro Arg Tyr 180 185 190 Ala Thr Leu Glu
His Pro Phe His Cys Pro Arg Val Leu Lys Val Pro 195 200 205 Ser Tyr
Leu Ser Tyr Lys Phe Leu Gly Glu Arg Asp Cys Ala Ala Pro 210 215 220
Cys Glu Pro Ala Arg Pro Asp Gly Ser Met Phe Phe Ser Gln Glu Glu 225
230 235 240 Thr Arg Phe Ala Arg Leu Trp Ile Leu Thr Trp Ser Val Leu
Cys Cys 245 250 255 Ala Ser Thr Phe Phe Thr Val Thr Thr Tyr Leu Val
Asp Met Gln Arg 260 265 270 Phe Arg Tyr Pro Glu Arg Pro Ile Ile Phe
Leu Ser Gly Cys Tyr Thr 275 280 285 Met Val Ser Val Ala Tyr Ile Ala
Gly Phe Val Leu Gln Glu Arg Val 290 295 300 Val Cys Asn Glu Arg Phe
Ser Glu Asp Gly Tyr Arg Thr Val Val Gln 305 310 315 320 Gly Thr Lys
Lys Glu Gly Cys Thr Ile Leu Phe Met Met Leu Tyr Phe 325 330 335 Phe
Ser Met Ala Ser Ser Ile Trp Trp Val Ile Leu Ser Leu Thr Trp 340 345
350 Phe Leu Ala Ala Gly Met Lys Trp Gly His Glu Ala Ile Glu Ala Asn
355 360 365 Ser Gln Tyr Phe His Leu Ala Ala Trp Ala Val Pro Ala Val
Lys Thr 370 375 380 Ile Thr Ile Leu Ala Met Gly Gln Ile Asp Gly Asp
Leu Leu Ser Gly 385 390 395 400 Val Cys Phe Val Gly Leu Asn Ser Leu
Asp Pro Leu Arg Gly Phe Val 405 410 415 Leu Ala Pro Leu Phe Val Tyr
Leu Phe Ile Gly Thr Ser Phe Leu Leu 420 425 430 Ala Gly Phe Val Ser
Leu Phe Arg Ile Arg Thr Ile Met Lys His Asp 435 440 445 Gly Thr Lys
Thr Glu Lys Leu Glu Arg Leu Met Val Arg Ile Gly Val 450 455 460 Phe
Ser Val Leu Tyr Thr Val Pro Ala Thr Ile Val Ile Ala Cys Tyr 465 470
475 480 Phe Tyr Glu Gln Ala Phe Arg Glu His Trp Glu Arg Ser Trp Val
Ser 485 490 495 Gln His Cys Lys Ser Leu Ala Ile Pro Cys Pro Ala His
Tyr Thr Pro 500 505 510 Arg Met Ser Pro Asp Phe Thr Val Tyr Met Ile
Lys Tyr Leu Met Thr 515 520 525 Leu Ile Val Gly Ile Thr Ser Gly Phe
Trp Ile Trp Ser Gly Lys Thr 530 535 540 Leu His Ser Trp Arg Lys Phe
Tyr Thr Arg Leu Thr Asn Ser Arg His 545 550 555 560 Gly Glu Thr Thr
Val 565 42 1983 DNA Homo sapiens human frizzled2 (Fzd2) 42
cgagtaaagt ttgcaaagag gcgcgggagg cggcagccgc agcgaggagg cggcggggaa
60 gaagcgcagt ctccgggttg ggggcggggg cggggggggc gccaaggagc
cgggtggggg 120 gcggcggcca gcatgcggcc ccgcagcgcc ctgccccgcc
tgctgctgcc gctgctgctg 180 ctgcccgccg ccgggccggc ccagttccac
ggggagaagg gcatctccat cccggaccac 240 ggcttctgcc agcccatctc
catcccgctg tgcacggaca tcgcctacaa ccagaccatc 300 atgcccaacc
ttctgggcca cacgaaccag gaggacgcag gcctagaggt gcaccagttc 360
tatccgctgg tgaaggtgca gtgctcgccc gaactgcgct tcttcctgtg ctccatgtac
420 gcacccgtgt gcaccgtgct ggaacaggcc atcccgccgt gccgctctat
ctgtgagcgc 480 gcgcgccagg gctgcgaagc cctcatgaac aagttcggtt
ttcagtggcc cgagcgcctg 540 cgctgcgagc acttcccgcg ccacggcgcc
gagcagatct gcgtcggcca gaaccactcc 600 gaggacggag ctcccgcgct
actcaccacc gcgccgccgc cgggactgca gccgggtgcc 660 gggggcaccc
cgggtggccc gggcggcggc ggcgctcccc cgcgctacgc cacgctggag 720
caccccttcc actgcccgcg cgtcctcaag gtgccatcct atctcagcta caagtttctg
780 ggcgagcgtg attgtgctgc gccctgcgaa cctgcgcggc ccgatggttc
catgttcttc 840 tcacaggagg agacgcgttt cgcgcgcctc tggatcctca
cctggtcggt gctgtgctgc 900 gcttccacct tcttcactgt caccacgtac
ttggtagaca tgcagcgctt ccgctaccca 960 gagcggccta tcatttttct
gtcgggctgc tacaccatgg tgtcggtggc ctacatcgcg 1020 ggcttcgtgc
tccaggagcg cgtggtgtgc aacgagcgct tctccgagga cggttaccgc 1080
acggtggtgc agggcaccaa gaaggagggc tgcaccatcc tcttcatgat gctctacttc
1140 ttcagcatgg ccagctccat ctggtgggtc atcctgtcgc tcacctggtt
cctggcagcc 1200 ggcatgaagt ggggccacga ggccatcgag gccaactctc
agtacttcca cctggccgcc 1260 tgggccgtgc cggccgtcaa gaccatcacc
atcctggcca tgggccagat cgacggcgac 1320 ctgctgagcg gcgtgtgctt
cgtaggcctc aacagcctgg acccgctgcg gggcttcgtg 1380 ctagcgccgc
tcttcgtgta cctgttcatc ggcacgtcct tcctcctggc cggcttcgtg 1440
tcgctcttcc gcatccgcac catcatgaag cacgacggca ccaagaccga aaagctggag
1500 cggctcatgg tgcgcatcgg cgtcttctcc gtgctctaca cagtgcccgc
caccatcgtc 1560 atcgcttgct acttctacga gcaggccttc cgcgagcact
gggagcgctc gtgggtgagc 1620 cagcactgca agagcctggc catcccgtgc
ccggcgcact acacgccgcg catgtcgccc 1680 gacttcacgg tctacatgat
caaatacctc atgacgctca tcgtgggcat cacgtcgggc 1740 ttctggatct
ggtcgggcaa gacgctgcac tcgtggagga agttctacac tcgcctcacc 1800
aacagccgac acggtgagac caccgtgtga gggacgcccc caggccggaa ccgcgcggcg
1860 ctttcctccg cccggggtgg ggcccctaca gactccgtat tttatttttt
taaataaaaa 1920 acgatcgaaa ccatttcact tttaggttgc tttttaaaag
agaactctct gcccaacacc 1980 ccc 1983 43 666 PRT Homo sapiens human
frizzled3 (Fzd3) 43 Met Ala Met Thr Trp Ile Val Phe Ser Leu Trp Pro
Leu Thr Val Phe 1 5 10 15 Met Gly His Ile Gly Gly His Ser Leu Phe
Ser Cys Glu Pro Ile Thr 20 25 30 Leu Arg Met Cys Gln Asp Leu Pro
Tyr Asn Thr Thr Phe Met Pro Asn 35 40 45 Leu Leu Asn His Tyr Asp
Gln Gln Thr Ala Ala Leu Ala Met Glu Pro 50 55 60 Phe His Pro Met
Val Asn Leu Asp Cys Ser Arg Asp Phe Arg Pro Phe 65 70 75 80 Leu Cys
Ala Leu Tyr Ala Pro Ile Cys Met Glu Tyr Gly Arg Val Thr 85 90 95
Leu Pro Cys Arg Arg Leu Cys Gln Arg Ala Tyr Ser Glu Cys Ser Lys 100
105 110 Leu Met Glu Met Phe Gly Val Pro Trp Pro Glu Asp Met Glu Cys
Ser 115 120 125 Arg Phe Pro Asp Cys Asp Glu Pro Tyr Pro Arg Leu Val
Asp Leu Asn 130 135 140 Leu Ala Gly Glu Pro Thr Glu Gly Ala Pro Val
Ala Val Gln Arg Asp 145 150 155 160 Tyr Gly Phe Trp Cys Pro Arg Glu
Leu Lys Ile Asp Pro Asp Leu Gly 165 170 175 Tyr Ser Phe Leu His Val
Arg Asp Cys Ser Pro Pro Cys Pro Asn Met 180 185 190 Tyr Phe Arg Arg
Glu Glu Leu Ser Phe Ala Arg Tyr Phe Ile Gly Leu 195 200 205 Ile Ser
Ile Ile Cys Leu Ser Ala Thr Leu Phe Thr Phe Leu Thr Phe 210 215 220
Leu Ile Asp Val Thr Arg Phe Arg Tyr Pro Glu Arg Pro Ile Ile Phe 225
230 235 240 Tyr Ala Val Cys Tyr Met Met Val Ser Leu Ile Phe Phe Ile
Gly Phe 245 250 255 Leu Leu Glu Asp Arg Val Ala Cys Asn Ala Ser Ile
Pro Ala Gln Tyr 260 265 270 Lys Ala Ser Thr Val Thr Gln Gly Ser His
Asn Lys Ala Cys Thr Met 275 280 285 Leu Phe Met Ile Leu Tyr Phe Phe
Thr Met Ala Gly Ser Val Trp Trp 290 295 300 Val Ile Leu Thr Ile Thr
Trp Phe Leu Ala Ala Val Pro Lys Trp Gly 305 310 315 320 Ser Glu Ala
Ile Glu Lys Lys Ala Leu Leu Phe His Ala Ser Ala Trp 325 330 335 Gly
Ile Pro Gly Thr Leu Thr Ile Ile Leu Leu Ala Met Asn Lys Ile 340 345
350 Glu Gly Asp Asn Ile Ser Gly Val Cys Phe Val Gly Leu Tyr Asp Val
355 360 365 Asp Ala Leu Arg Tyr Phe Val Leu Ala Pro Leu Cys Leu Tyr
Val Val 370 375 380 Val Gly Val Ser Leu Leu Leu Ala Gly Ile Ile Ser
Leu Asn Arg Val 385 390 395 400 Arg Ile Glu Ile Pro Leu Glu Lys Glu
Asn Gln Asp Lys Leu Val Lys 405 410 415 Phe Met Ile Arg Ile Gly Val
Phe Ser Ile Leu Tyr Leu Val Pro Leu 420 425 430 Leu Val Val Ile Gly
Cys Tyr Phe Tyr Glu Gln Ala Tyr Arg Gly Ile 435 440 445 Trp Glu Thr
Thr Trp Ile Gln Glu Arg Cys Arg Glu Tyr His Ile Pro 450 455 460 Cys
Pro Tyr Gln Val Thr Gln Met Ser Arg Pro Asp Leu Ile Leu Phe 465 470
475 480 Leu Met Lys Tyr Leu Met Ala Leu Ile Val Gly Ile Pro Ser Val
Phe 485 490 495 Trp Val Gly Ser Lys Lys Thr Cys Phe Glu Trp Ala Ser
Phe Phe His 500 505 510 Gly Arg Arg Lys Lys Glu Ile Val Asn Glu Ser
Arg Gln Val Leu Gln 515 520 525 Glu Pro Asp Phe Ala Gln Ser Leu Leu
Arg Asp Pro Asn Thr Pro Ile 530 535 540 Ile Arg Lys Ser Arg Gly Thr
Ser Thr Gln Gly Thr Ser Thr His Ala 545 550 555 560 Ser Ser Thr Gln
Leu Ala Met Val Asp Asp Gln Arg Ser Lys Ala Gly 565 570 575 Ser Ile
His Ser Lys Val Ser Ser Tyr His Gly Ser Leu His Arg Ser 580 585 590
Arg Asp Gly Arg Tyr Thr Pro Cys Ser Tyr Arg Gly Met Glu Glu Arg 595
600 605 Leu Pro His Gly Ser Met Ser Arg Leu Thr Asp His Ser Arg His
Ser 610 615 620 Ser Ser His Arg Leu Asn Glu Gln Ser Arg His Ser Ser
Ile Arg Asp 625 630 635 640 Leu Ser Asn Asn Pro Met Thr His Ile Thr
His Gly Thr Ser Met Asn 645 650 655 Arg Val Ile Glu Glu Asp Gly Thr
Ser Ala 660 665 44 3933 DNA Homo sapiens human frizzled3 (Fzd3) 44
gccgctccgg gtacctgagg gacgcgcggc cgcccgcggc aggcggtgca gcccccccac
60 cccttggagc caggcgccgg ggtctgagga tagcatttct caagacctga
cttatggagc 120 acttgtaacc tgagatattt cagttgaagg aagaaatagc
tcttctccta agatggaatc 180 tgtggtttgg gaatgtggtt gatcaacttg
atatgttggc caaatgtgcc ccatgtaata 240 aaatgaaaag aagagacaag
atgatgtcat tttcccatat tgtgaaacca aaaacaaacg 300 ccttttgtga
gaccaagcta acaaacctct gacggtgcga agagtattta actgtttgaa 360
gaatttaaca gtaagataca gaagaagtac cttcgagctg agacctgcag gtgtataaat
420 atctaaaata catattgaat aggcctgatc atctgaatct ccttcagacc
caggaaggat 480 ggctatgact tggattgtct tctctctttg gcccttgact
gtgttcatgg ggcatatagg 540 tgggcacagt ttgttttctt gtgaacctat
taccttgagg atgtgccaag atttgcctta 600 taatactacc ttcatgccta
atcttctgaa tcattatgac caacagacag cagctttggc 660 aatggagcca
ttccacccta tggtgaatct ggattgttct cgggatttcc ggccttttct 720
ttgtgcactc tacgctccta tttgtatgga atatggacgt gtcacacttc cctgtcgtag
780 gctgtgtcag cgggcttaca gtgagtgttc gaagctcatg gagatgtttg
gtgttccttg 840 gcctgaagat atggaatgca gtaggttccc agattgtgat
gagccatatc ctcgacttgt 900 ggatctgaat ttagctggag aaccaactga
aggagcccca gtggcagtgc agagagacta 960 tggtttttgg tgtccccgag
agttaaaaat tgatcctgat ctgggttatt cttttctgca 1020 tgtgcgtgat
tgttcacctc cttgtccaaa tatgtacttc agaagagaag aactgtcatt 1080
tgctcgctat ttcataggat tgatttcaat catttgcctc tcggccacat tgtttacttt
1140 tttaactttt ttgattgatg tcacaagatt ccgttatcct gaaaggccta
ttatatttta 1200 tgcagtctgc tacatgatgg tatccttaat tttcttcatt
ggatttttgc ttgaagatcg 1260 agtagcctgc aatgcatcca tccctgcaca
atataaggct tccacagtga cacaaggatc 1320 tcataataaa gcctgtacca
tgctttttat gatactctat ttttttacta tggctggcag 1380 tgtatggtgg
gtaattctta ccatcacatg gtttttagca gctgtgccaa agtggggtag 1440
tgaagctatt gagaagaaag cattgctgtt tcacgccagt gcatggggca tccccggaac
1500 tctaaccatc atccttttag cgatgaataa aattgaaggt gacaatatta
gtggcgtgtg 1560 ttttgttggc ctctacgatg ttgatgcatt gagatatttt
gttcttgctc ccctctgcct 1620 gtatgtggta gttggggttt ctctcctctt
agctggcatt atatccctaa acagagttcg 1680 aattgagatt ccattagaaa
aggagaacca agataaatta gtgaagttta tgatccggat 1740 cggtgttttc
agcattcttt atctcgtacc actcttggtt gtaattggat gctactttta 1800
tgagcaagct taccggggca tctgggaaac aacgtggata caagaacgct gcagagaata
1860 tcacattcca tgtccatatc aggttactca aatgagtcgt ccagacttga
ttctctttct 1920 gatgaaatac ctgatggctc tcatagttgg cattccctct
gtattttggg ttggaagcaa
1980 aaagacatgc tttgaatggg ccagtttttt tcatggtcgt aggaaaaaag
agatagtgaa 2040 tgagagccga caggtactcc aggaacctga ttttgctcag
tctctcctga gggatccaaa 2100 tactcctatc ataagaaagt caaggggaac
ttccactcaa ggaacatcca cccatgcttc 2160 ttcaactcag ctggctatgg
tggatgatca aagaagcaaa gcaggaagca tccacagcaa 2220 agtgagcagc
taccacggca gcctccacag atcacgtgat ggcaggtaca cgccctgcag 2280
ttacagagga atggaggaga gactacctca tggcagcatg tcacgactaa cagatcactc
2340 caggcatagt agttctcatc ggctcaatga acagtcacga catagcagca
tcagagatct 2400 cagtaataat cccatgactc atatcacaca tggcaccagc
atgaatcggg ttattgaaga 2460 agatggaacc agtgcttaat ttgtcttgtc
taaggtggaa atcttgtgct gtttaaaaag 2520 cagattttat tctttgcctt
ttgcatgact gatagctgta actcacagtt aacatgcttt 2580 cagtcaagta
cagattgtgt ccactggaaa ggtaaatgat tgctttttta tattgcatca 2640
aacttggaac atcaaggcat ccaaaacact aagaattcta tcatcacaaa aataattcgt
2700 ctttctaggt tatgaagaga taattatttg tctggtaagc atttttataa
acccactcat 2760 tttatattta gaaaaatcct aaatgtgtgg tgactgcttt
gtagtgaact ttcatatact 2820 ataaactagt tgtgagataa cattctggta
gctcagttaa taaaacaatt tcagaattaa 2880 agaaattttc tatgcaaggt
ttacttctca gatgaacagt aggactttgt agttttattt 2940 ccactaagtg
aaaaaagaac tgtgttttta aactgtagga gaatttaata aatcagcaag 3000
ggtattttag ctaatagaat aaaagtgcaa cagaagaatt tgattagtct atgaaaggtt
3060 ctcttaaaat tctatcgaaa taatcttcat gcagagatat tcagggtttg
gattagcagt 3120 ggaataaaga gatgggcatt gtttccccta taattgtgct
gtttttataa cttttgtaaa 3180 tattactttt tctggctgtg tttttataac
ttatccatat gcatgatgga aaaattttaa 3240 tttgtagcca tcttttccca
tgtaatagta ttgattcata gagaacttaa tgttcaaaat 3300 ttgctttgtg
gaggcatgta ataagataaa catcatacat tataaggtaa ccacaattac 3360
aaaatggcaa aacattttct ctgtattcat tgttgtattt ttctacagtg agatgtgatc
3420 ttgccaaagc caccagacct tggcttccag gccctcctgt agtgagttga
ttgtctgcac 3480 ttgccttgcc caatagccag taggctacag cttttgcccc
acacccttat tttcagattc 3540 tggatcattc ttgtttacaa ctgaaatata
tataacctca gtccaaagtg gtgattgatt 3600 tgagtatttg aaaattgttg
tagctaaatg aagcatgatt agtcttagta tgaatatcat 3660 ttaatcttta
aaaaatcaag taaaaatgtt tatctgataa tgtttaaata atttacaata 3720
taaactgtaa aacttattag gcatgaaatc aatcagaaga gaaagaaaaa tgctggaaca
3780 tgcttgatgt attatgtaaa aagcatattt aaacaagggt cctcaaccct
gactgcagat 3840 aagaatcact tgggttactt cagatgccta acaccttcct
ctcatacaaa taagaattgg 3900 tagctttctt aaaaaaaaaa aaaaaaaaaa aaa
3933 45 537 PRT Homo sapiens human frizzled4 (Fzd4) 45 Met Ala Trp
Arg Gly Ala Gly Pro Ser Val Pro Gly Ala Pro Gly Gly 1 5 10 15 Val
Gly Leu Ser Leu Gly Leu Leu Leu Gln Leu Leu Leu Leu Leu Gly 20 25
30 Pro Ala Arg Gly Phe Gly Asp Glu Glu Glu Arg Arg Cys Asp Pro Ile
35 40 45 Arg Ile Ser Met Cys Gln Asn Leu Gly Tyr Asn Val Thr Lys
Met Pro 50 55 60 Asn Leu Val Gly His Glu Leu Gln Thr Asp Ala Glu
Leu Gln Leu Thr 65 70 75 80 Thr Phe Thr Pro Leu Ile Gln Tyr Gly Cys
Ser Ser Gln Leu Gln Phe 85 90 95 Phe Leu Cys Ser Val Tyr Val Pro
Met Cys Thr Glu Lys Ile Asn Ile 100 105 110 Pro Ile Gly Pro Cys Gly
Gly Met Cys Leu Ser Val Lys Arg Arg Cys 115 120 125 Glu Pro Val Leu
Lys Glu Phe Gly Phe Ala Trp Pro Glu Ser Leu Asn 130 135 140 Cys Ser
Lys Phe Pro Pro Gln Asn Asp His Asn His Met Cys Met Glu 145 150 155
160 Gly Pro Gly Asp Glu Glu Val Pro Leu Pro His Lys Thr Pro Ile Gln
165 170 175 Pro Gly Glu Glu Cys His Ser Val Gly Thr Asn Ser Asp Gln
Tyr Ile 180 185 190 Trp Val Lys Arg Ser Leu Asn Cys Val Leu Lys Cys
Gly Tyr Asp Ala 195 200 205 Gly Leu Tyr Ser Arg Ser Ala Lys Glu Phe
Thr Asp Ile Trp Met Ala 210 215 220 Val Trp Ala Ser Leu Cys Phe Ile
Ser Thr Ala Phe Thr Val Leu Thr 225 230 235 240 Phe Leu Ile Asp Ser
Ser Arg Phe Ser Tyr Pro Glu Arg Pro Ile Ile 245 250 255 Phe Leu Ser
Met Cys Tyr Asn Ile Tyr Ser Ile Ala Tyr Ile Val Arg 260 265 270 Leu
Thr Val Gly Arg Glu Arg Ile Ser Cys Asp Phe Glu Glu Ala Ala 275 280
285 Glu Pro Val Leu Ile Gln Glu Gly Leu Lys Asn Thr Gly Cys Ala Ile
290 295 300 Ile Phe Leu Leu Met Tyr Phe Phe Gly Met Ala Ser Ser Ile
Trp Trp 305 310 315 320 Val Ile Leu Thr Leu Thr Trp Phe Leu Ala Ala
Gly Leu Lys Trp Gly 325 330 335 His Glu Ala Ile Glu Met His Ser Ser
Tyr Phe His Ile Ala Ala Trp 340 345 350 Ala Ile Pro Ala Val Lys Thr
Ile Val Ile Leu Ile Met Arg Leu Val 355 360 365 Asp Ala Asp Glu Leu
Thr Gly Leu Cys Tyr Val Gly Asn Gln Asn Leu 370 375 380 Asp Ala Leu
Thr Gly Phe Val Val Ala Pro Leu Phe Thr Tyr Leu Val 385 390 395 400
Ile Gly Thr Leu Phe Ile Ala Ala Gly Leu Val Ala Leu Phe Lys Ile 405
410 415 Arg Ser Asn Leu Gln Lys Asp Gly Thr Lys Thr Asp Lys Leu Glu
Arg 420 425 430 Leu Met Val Lys Ile Gly Val Phe Ser Val Leu Tyr Thr
Val Pro Ala 435 440 445 Thr Cys Val Ile Ala Cys Tyr Phe Tyr Glu Ile
Ser Asn Trp Ala Leu 450 455 460 Phe Arg Tyr Ser Ala Asp Asp Ser Asn
Met Ala Val Glu Met Leu Lys 465 470 475 480 Ile Phe Met Ser Leu Leu
Val Gly Ile Thr Ser Gly Met Trp Ile Trp 485 490 495 Ser Ala Lys Thr
Leu His Thr Trp Gln Lys Cys Ser Asn Arg Leu Val 500 505 510 Asn Ser
Gly Lys Val Lys Arg Glu Lys Arg Gly Asn Gly Trp Val Lys 515 520 525
Pro Gly Lys Gly Ser Glu Thr Val Val 530 535 46 7391 DNA Homo
sapiens human frizzled4 (Fzd4) 46 gctgcgcagc gctggctgct ggctggcctc
gcggagacgc cgaacggacg cggccggcgc 60 cggcttgtgg gctcgccgcc
tgcagccatg accctcgcag cctgtccctc ggcctcggcc 120 cgggacgtct
aaaatcccac acagtcgcgc gcagctgctg gagagccggc cgctgccccc 180
tcgtcgccgc atcacactcc cgtcccggga gctgggagca gcgcgggcag ccggcgcccc
240 cgtgcaaact gggggtgtct gccagagcag ccccagccgc tgccgctgct
acccccgatg 300 ctggccatgg cctggcgggg cgcagggccg agcgtcccgg
gggcgcccgg gggcgtcggt 360 ctcagtctgg ggttgctcct gcagttgctg
ctgctcctgg ggccggcgcg gggcttcggg 420 gacgaggaag agcggcgctg
cgaccccatc cgcatctcca tgtgccagaa cctcggctac 480 aacgtgacca
agatgcccaa cctggttggg cacgagctgc agacggacgc cgagctgcag 540
ctgacaactt tcacaccgct catccagtac ggctgctcca gccagctgca gttcttcctt
600 tgttctgttt atgtgccaat gtgcacagag aagatcaaca tccccattgg
cccatgcggc 660 ggcatgtgtc tttcagtcaa gagacgctgt gaacccgtcc
tgaaggaatt tggatttgcc 720 tggccagaga gtctgaactg cagcaaattc
ccaccacaga acgaccacaa ccacatgtgc 780 atggaagggc caggtgatga
agaggtgccc ttacctcaca aaacccccat ccagcctggg 840 gaagagtgtc
actctgtggg aaccaattct gatcagtaca tctgggtgaa aaggagcctg 900
aactgtgtgc tcaagtgtgg ctatgatgct ggcttataca gccgctcagc caaggagttc
960 actgatatct ggatggctgt gtgggccagc ctgtgtttca tctccactgc
cttcacagta 1020 ctgaccttcc tgatcgattc ttctaggttt tcctaccctg
agcgccccat catatttctc 1080 agtatgtgct ataatattta tagcattgct
tatattgtca ggctgactgt aggccgggaa 1140 aggatatcct gtgattttga
agaggcagca gaacctgttc tcatccaaga aggacttaag 1200 aacacaggat
gtgcaataat tttcttgctg atgtactttt ttggaatggc cagctccatt 1260
tggtgggtta ttctgacact cacttggttt ttggcagcag gactcaaatg gggtcatgaa
1320 gccattgaaa tgcacagctc ttatttccac attgcagcct gggccatccc
cgcagtgaaa 1380 accattgtca tcttgattat gagactggtg gatgcagatg
aactgactgg cttgtgctat 1440 gttggaaacc aaaatctcga tgccctcacc
gggttcgtgg tggctcccct ctttacttat 1500 ttggtcattg gaactttgtt
cattgctgca ggtttggtgg ccttgttcaa aattcggtca 1560 aatcttcaaa
aggatgggac aaagacagac aagttagaaa gactgatggt caagattggg 1620
gtgttctcag tactgtacac agttcctgca acgtgtgtga ttgcctgtta tttttatgaa
1680 atctccaact gggcactttt tcggtattct gcagatgatt ccaacatggc
tgttgaaatg 1740 ttgaaaattt ttatgtcttt gttggtgggc atcacttcag
gcatgtggat ttggtctgcc 1800 aaaactcttc acacgtggca gaagtgttcc
aacagattgg tgaattctgg aaaggtaaag 1860 agagagaaga gaggaaatgg
ttgggtgaag cctggaaaag gcagtgagac tgtggtataa 1920 ggctagtcag
cctccatgct ttcttcattt tgaagggggg aatgccagca ttttggagga 1980
aattctacta aaagttttat gcagtgaatc tcagtttgaa caaactagca acaattaagt
2040 gacccccgtc aacccactgc ctcccacccc gaccccagca tcaaaaaacc
aatgattttg 2100 ctgcagactt tggaatgatc caaaatggaa aagccagtta
gaggctttca aagctgtgaa 2160 aaatcaaaac gttgatcact ttagcaggtt
gcagcttgga gcgtggaggt cctgcctaga 2220 ttccaggaag tccagggcga
tactgttttc ccctgcaggg tgggatttga gctgtgagtt 2280 ggtaactagc
agggagaaat attaactttt ttaacccttt accattttaa atactaactg 2340
ggtctttcag atagcaaagc aatctataaa cactggaaac gctgggttca gaaaagtgtt
2400 acaagagttt tatagtttgg ctgatgtaac ataaacatct tctgtggtgc
gctgtctgct 2460 gtttagaact ttgtggactg cactcccaag aagtggtgtt
agaatctttc agtgcctttg 2520 tcataaaaca gttatttgaa caaacaaaag
tactgtactc acacacataa ggtatccagt 2580 ggatttttct tctctgtctt
cctctcttaa atttcaacat ctctcttctt ggctgctgct 2640 gttttcttca
ttttatgtta atgactcaaa aaaggtattt ttatagaatt tttgtactgc 2700
agcatgctta aagaggggaa aaggaagggt gattcacttt ctgacaatca cttaattcag
2760 aggaaaatga gatttactaa gttgacttac ctgacggacc ccagagacct
attgcattga 2820 gcagtgggga cttaatatat tttacttgtg tgattgcatc
tatgcagacg ccagtctgga 2880 agagctgaaa tgttaagttt cttggcaact
ttgcattcac acagattagc tgtgtaattt 2940 ttgtgtgtca attacaatta
aaagcacatt gttggaccat gacatagtat actcaactga 3000 ctttaaaact
atggtcaact tcaacttgca ttctcagaat gatagtgcct ttaaaaattt 3060
ttttattttt taaagcataa gaatgttatc agaatctggt ctacttagga caatggagac
3120 tttttcagtt ttataaaggg aactgaggac agctaatcca actacttggt
gcgtaattgt 3180 ttcctagtaa ttggcaaagg ctccttgtaa gatttcactg
gaggcagtgt ggcctggagt 3240 atttatatgg tgcttaatga atctccagaa
tgccagccag aagcctgatt ggttagtagg 3300 gaataaagtg tagaccatat
gaaatgaact gcaaactcta atagcccagg tcttaattgc 3360 ctttagcaga
ggtatccaaa gcttttaaaa tttatgcata cgttcttcac aagggggtac 3420
ccccagcagc ctctcgaaaa ttgcacttct cttaaaactg taactggcct ttctcttacc
3480 ttgccttagg ccttctaatc atgagatctt ggggacaaat tgactatgtc
acaggttgct 3540 ctccttgtaa ctcatacctg tctgcttcag caactgcttt
gcaatgacat ttatttatta 3600 attcatgcct taaaaaaata ggaagggaag
cttttttttt tctttttttt tttttcaatc 3660 acactttgtg gaaaaacatt
tccagggact caaaattcca aaaaggtggt caaattctgg 3720 aagtaagcat
ttcctctttt ttaaaaattt ggtttgagcc ttatgcccat agtttgacat 3780
ttccctttct tctttccttt ttgtttttgt gtggttcttg agctctctga catcaagatg
3840 catgtaaagt cgattgtatg ttttggaagg caaagtcttg gcttttgaga
ctgaagttaa 3900 gtgggcacag gtggcccctg ctgctgtgcc cagtctgagt
accttggcta gactctaggt 3960 caggctccag gagcatgaga attgatcccc
agaagaacca ttttaactcc atctgatact 4020 ccattgccta tgaaatgtaa
aatgtgaact ccctgtgctg cttgtagaca gttcccataa 4080 ctgtccacgg
ccctggagca cgcacccagg ggcagagcct gcccttactc acgctctgct 4140
ctggtgtctt gggagttgtg cagggactct ggcccaggca ggggaaggaa gaccaggcgg
4200 taggggactg gtcttgctgt tagagtatag aggtttgtaa tgcagttttc
ttcataatgt 4260 gtcagtgatt gtgtgaccaa ggcagcatct agcagaaagc
caggcatgga gtaggtgatc 4320 gatacttgtc aatgactaaa taataacaat
aaaagagcac ttgggtgaat ctgggcacct 4380 gatttctgag ttttgagttc
tggagctagt gttttgacaa tgctttgggt tttgacatgc 4440 cttttccaca
aatctcttgc cttttcaggg caaagtgtat ttgatcagaa gtggccattt 4500
ggattagtag ccttagcaat gctacagggt tataggcctc tcctttcaca ttccagacaa
4560 tggagagtgt ttatggtttc aggaaaagaa ctttgtggct gaggggtcag
ttaccagtga 4620 ccttcaatca actccatcac ttcttaaatc ggtatttgtt
aaaaaaatca gttattttat 4680 ttattgagtg ccgactgtag taaagccctg
aaatagataa tctctgttct tctaactgat 4740 ctaggatggg gacgcaccca
ggtctgctga actttactgt tcctctggga aaggagcagg 4800 gacctctgga
attcccatct gtttcactgt ctccattcca taaatctctt cctgtgtgag 4860
ccaccacacc cagcctgggt ctctctactt ttaacacatc tctcatccct ttcccaggat
4920 tccttccaag tcagttacag gtggttttaa cagaaagcat cagctctgct
tcgtgacagt 4980 ctctggagaa atcccttagg aagactatga gagtaggcca
caaggacatg ggcccacaca 5040 tctgctttgg ctttgccggc aattcagggc
ttggggtatt ccatgtgact tgtataggta 5100 tatttgagga cagcatcttg
ctagagaaaa ggtgagggtt gtttttcttt ctctgaaacc 5160 tacagtaaat
gggtatgatt gtagcttcct cagaaatccc ttggcctcca gagattaaac 5220
atggtgcaat ggcacctctg tccaacctcc tttctggtag attcctttct cctgcttcat
5280 ataggccaaa cctcagggca agggaacatg ggggtagagt ggtgctggcc
agaaccatct 5340 gcttgagcta cttggttgat tcatatcctc tttcctttat
ggagacccat ttcctgatct 5400 ctgagactgt tgctgaactg gcaacttact
tgggcctgaa actggagaag gggtgacatt 5460 tttttaattt cagagatgct
ttctgatttt cctctcccag gtcactgtct cacctgcact 5520 ctccaaactc
aggttccggg aagcttgtgt gtctagatac tgaattgaga ttctgttcag 5580
caccttttag ctctatactc tctggctccc ctcatcctca tggtcactga attaaatgct
5640 tattgtattg agaaccaaga tgggacctga ggacacaaag atgagctcaa
cagtctcagc 5700 cctagaggaa tagactcagg gatttcacca ggtcggtgca
gtatttgatt tctggtgagg 5760 tgaccacagc tgcagttagg gaagggagcc
attgagcaca gactttggaa ggaacctttt 5820 ttttgttgtt tgtttgtttg
tttgtttgtt tgtttgtttg agacagggtc ttgctctgtc 5880 acccaggctg
gggcgcaatg gcacgatctt ggctcactgc aacctctgcc tcctgggttc 5940
aagtgattct cctgccacag cctcctgagg agctgggact acaggtgcgt gctaccacgc
6000 ccagctactt ctgtattttt agtagagacg gggtttcact gtgttggcca
ggctggtctc 6060 gaactcctga cctcatgatc tgcccgcctc agcctcccaa
agtgctggga ttacaagtgt 6120 gagccaccac acctggcctg gaaggaacct
cttaaaatca gtttacgtct tgtattttgt 6180 tctgtgatgg aggacactgg
agagagttgc tattccagtc aatcatgtcg agtcactgga 6240 ctctgaaaat
cctattggtt cctttatttt atttgagttt agagttccct tctgggtttg 6300
tattatgtct ggcaaatgac ctgggttatc acttttcctc cagggttaga tcatagatct
6360 tggaaactcc ttagagagca ttttgctcct accaaggatc agatactgga
gccccacata 6420 atagatttca tttcactcta gcctacatag agctttctgt
tgctgtctct tgccatgcac 6480 ttgtgcggtg attacacact tgacagtacc
aggagacaaa tgacttacag atcccccgac 6540 atgcctcttc cccttggcaa
gctcagttgc cctgatagta gcatgtttct gtttctgatg 6600 tacctttttt
ctcttcttct ttgcatcagc caattcccag aatttcccca ggcaatttgt 6660
agaggacctt tttggggtcc tatatgagcc atgtcctcaa agcttttaaa cctccttgct
6720 ctcctacaat attcagtaca tgaccactgt catcctagaa ggcttctgaa
aagaggggca 6780 agagccactc tgcgccacaa aggttgggtc catcttctct
ccgaggttgt gaaagttttc 6840 aaattgtact aataggctgg ggccctgact
tggctgtggg ctttgggagg ggtaagctgc 6900 tttctagatc tctcccagtg
aggcatggag gtgtttctga attttgtcta cctcacaggg 6960 atgttgtgag
gcttgaaaag gtcaaaaaat gatggcccct tgagctcttt gtaagaaagg 7020
tagatgaaat atcggatgta atctgaaaaa aagataaaat gtgacttccc ctgctctgtg
7080 cagcagtcgg gctggatgct ctgtggcctt tcttgggtcc tcatgccacc
ccacagctcc 7140 aggaaccttg aagccaatct gggggacttt cagatgtttg
acaaagaggt accaggcaaa 7200 cttcctgcta cacatgccct gaatgaattg
ctaaatttca aaggaaatgg accctgcttt 7260 taaggatgta caaaagtatg
tctgcatcga tgtctgtact gtaaatttct aatttatcac 7320 tgtacaaaga
aaaccccttg ctatttaatt ttgtattaaa ggaaaataaa gttttgtttg 7380
ttaaaaaaaa a 7391 47 585 PRT Homo sapiens human frizzled5 (Fzd5) 47
Met Ala Arg Pro Asp Pro Ser Ala Pro Pro Ser Leu Leu Leu Leu Leu 1 5
10 15 Leu Ala Gln Leu Val Gly Arg Ala Ala Ala Ala Ser Lys Ala Pro
Val 20 25 30 Cys Gln Glu Ile Thr Val Pro Met Cys Arg Gly Ile Gly
Tyr Asn Leu 35 40 45 Thr His Met Pro Asn Gln Phe Asn His Asp Thr
Gln Asp Glu Ala Gly 50 55 60 Leu Glu Val His Gln Phe Trp Pro Leu
Val Glu Ile Gln Cys Ser Pro 65 70 75 80 Asp Leu Arg Phe Phe Leu Cys
Thr Met Tyr Thr Pro Ile Cys Leu Pro 85 90 95 Asp Tyr His Lys Pro
Leu Pro Pro Cys Arg Ser Val Cys Glu Arg Ala 100 105 110 Lys Ala Gly
Cys Ser Pro Leu Met Arg Gln Tyr Gly Phe Ala Trp Pro 115 120 125 Glu
Arg Met Ser Cys Asp Arg Leu Pro Val Leu Gly Arg Asp Ala Glu 130 135
140 Val Leu Cys Met Asp Tyr Asn Arg Ser Glu Ala Thr Thr Ala Pro Pro
145 150 155 160 Arg Pro Phe Pro Ala Lys Pro Thr Leu Pro Gly Pro Pro
Gly Ala Pro 165 170 175 Ala Ser Gly Gly Glu Cys Pro Ala Gly Gly Pro
Phe Val Cys Lys Cys 180 185 190 Arg Glu Pro Phe Val Pro Ile Leu Lys
Glu Ser His Pro Leu Tyr Asn 195 200 205 Lys Val Arg Thr Gly Gln Val
Pro Asn Cys Ala Val Pro Cys Tyr Gln 210 215 220 Pro Ser Phe Ser Ala
Asp Glu Arg Thr Phe Ala Thr Phe Trp Ile Gly 225 230 235 240 Leu Trp
Ser Val Leu Cys Phe Ile Ser Thr Ser Thr Thr Val Ala Thr 245 250 255
Phe Leu Ile Asp Met Asp Thr Phe Arg Tyr Pro Glu Arg Pro Ile Ile 260
265 270 Phe Leu Ser Ala Cys Tyr Leu Cys Val Ser Leu Gly Phe Leu Val
Arg 275 280 285 Leu Val Val Gly His Ala Ser Val Ala Cys Ser Arg Glu
His Asn His 290 295 300 Ile His Tyr Glu Thr Thr Gly Pro Ala Leu Cys
Thr Ile Val Phe Leu 305 310 315 320 Leu Val Tyr Phe Phe Gly Met Ala
Ser Ser Ile Trp Trp Val Ile Leu 325 330 335 Ser Leu Thr Trp Phe Leu
Ala Ala Ala Met Lys Trp Gly Asn Glu Ala 340 345 350 Ile Ala Gly Tyr
Gly Gln Tyr Phe His Leu Ala
Ala Trp Leu Ile Pro 355 360 365 Ser Val Lys Ser Ile Thr Ala Leu Ala
Leu Ser Ser Val Asp Gly Asp 370 375 380 Pro Val Ala Gly Ile Cys Tyr
Val Gly Asn Gln Asn Leu Asn Ser Leu 385 390 395 400 Arg Arg Phe Val
Leu Gly Pro Leu Val Leu Tyr Leu Leu Val Gly Thr 405 410 415 Leu Phe
Leu Leu Ala Gly Phe Val Ser Leu Phe Arg Ile Arg Ser Val 420 425 430
Ile Lys Gln Gly Gly Thr Lys Thr Asp Lys Leu Glu Lys Leu Met Ile 435
440 445 Arg Ile Gly Ile Phe Thr Leu Leu Tyr Thr Val Pro Ala Ser Ile
Val 450 455 460 Val Ala Cys Tyr Leu Tyr Glu Gln His Tyr Arg Glu Ser
Trp Glu Ala 465 470 475 480 Ala Leu Thr Cys Ala Cys Pro Gly His Asp
Thr Gly Gln Pro Arg Ala 485 490 495 Lys Pro Glu Tyr Trp Val Leu Met
Leu Lys Tyr Phe Met Cys Leu Val 500 505 510 Val Gly Ile Thr Ser Gly
Val Trp Ile Trp Ser Gly Lys Thr Val Glu 515 520 525 Ser Trp Arg Arg
Phe Thr Ser Arg Cys Cys Cys Arg Pro Arg Arg Gly 530 535 540 His Lys
Ser Gly Gly Ala Met Ala Ala Gly Asp Tyr Pro Glu Ala Ser 545 550 555
560 Ala Ala Leu Thr Gly Arg Thr Gly Pro Pro Gly Pro Ala Ala Thr Tyr
565 570 575 His Lys Gln Val Ser Leu Ser His Val 580 585 48 2334 DNA
Homo sapiens human frizzled5 (Fzd5) 48 acccagggac ggaggaccca
ggctggcttg gggactgtct gctcttctcg gcgggagccg 60 tggagagtcc
tttccctgga atccgagccc taaccgtctc tccccagccc tatccggcga 120
ggagcggagc gctgccagcg gaggcagcgc cttcccgaag cagtttatct ttggacggtt
180 ttctttaaag gaaaaacgaa ccaacaggtt gccagccccg gcgccacaca
cgagacgccg 240 gagggagaag ccccggcccg gattcctctg cctgtgtgcg
tccctcgcgg gctgctggag 300 gcgaggggag ggagggggcg atggctcggc
ctgacccatc cgcgccgccc tcgctgttgc 360 tgctgctcct ggcgcagctg
gtgggccggg cggccgccgc gtccaaggcc ccggtgtgcc 420 aggaaatcac
ggtgcccatg tgccgcggca tcggctacaa cctgacgcac atgcccaacc 480
agttcaacca cgacacgcag gacgaggcgg gcctggaggt gcaccagttc tggccgctgg
540 tggagatcca atgctcgccg gacctgcgct tcttcctatg cactatgtac
acgcccatct 600 gtctgcccga ctaccacaag ccgctgccgc cctgccgctc
ggtgtgcgag cgcgccaagg 660 ccggctgctc gccgctgatg cgccagtacg
gcttcgcctg gcccgagcgc atgagctgcg 720 accgcctccc ggtgctgggc
cgcgacgccg aggtcctctg catggattac aaccgcagcg 780 aggccaccac
ggcgcccccc aggcctttcc cagccaagcc cacccttcca ggcccgccag 840
gggcgccggc ctcggggggc gaatgccccg ctgggggccc gttcgtgtgc aagtgtcgcg
900 agcccttcgt gcccattctg aaggagtcac acccgctcta caacaaggtg
cggacgggcc 960 aggtgcccaa ctgcgcggta ccctgctacc agccgtcctt
cagtgccgac gagcgcacgt 1020 tcgccacctt ctggataggc ctgtggtcgg
tgctgtgctt catctccacg tccaccacag 1080 tggccacctt cctcatcgac
atggacacgt tccgctatcc tgagcgcccc atcatcttcc 1140 tgtcagcctg
ctacctgtgc gtgtcgctgg gcttcctggt gcgtctggtc gtgggccatg 1200
ccagcgtggc ctgcagccgc gagcacaacc acatccacta cgagaccacg ggccctgcac
1260 tgtgcaccat cgtcttcctc ctggtctact tcttcggcat ggccagctcc
atctggtggg 1320 tcatcctgtc gctcacctgg ttcctggccg ccgcgatgaa
gtggggcaac gaggccatcg 1380 cgggctacgg ccagtacttc cacctggctg
cgtggctcat ccccagcgtc aagtccatca 1440 cggcactggc gctgagctcc
gtggacgggg acccagtggc cggcatctgc tacgtgggca 1500 accagaacct
gaactcgctg cggcgcttcg tgctgggccc gctggtgctc tacctgctgg 1560
tgggcacgct cttcctgctg gcgggcttcg tgtcgctctt ccgcatccgc agcgtcatca
1620 agcagggcgg caccaagacg gacaagctgg agaagctcat gatccgcatc
ggcatcttca 1680 cgctgctcta cacggtcccc gccagcattg tggtggcctg
ctacctgtac gagcagcact 1740 accgcgagag ctgggaggcg gcgctcacct
gcgcctgccc gggccacgac accggccagc 1800 cgcgcgccaa gcccgagtac
tgggtgctca tgctcaagta cttcatgtgc ctggtggtgg 1860 gcatcacgtc
gggcgtctgg atctggtcgg gcaagacggt ggagtcgtgg cggcgtttca 1920
ccagccgctg ctgctgccgc ccgcggcgcg gccacaagag cgggggcgcc atggccgcag
1980 gggactaccc cgaggcgagc gccgcgctca caggcaggac cgggccgccg
ggccccgccg 2040 ccacctacca caagcaggtg tccctgtcgc acgtgtagga
ggctgccgcc gagggactcg 2100 gccggagagc tgaggggagg ggggcgtttt
gtttggtagt tttgccaagg tcacttccgt 2160 ttaccttcat ggtgctgttg
ccccctcccg cggcgacttg gagagaggga agaggggcgt 2220 tttcgaggaa
gaacctgtcc caggtcttct ccaaggggcc cagctcacgt gtattctatt 2280
ttgcgtttct tacctgcctt ctttatggga accctctttt taatttatat gtat 2334 49
706 PRT Homo sapiens human frizzled6 (Fzd6) 49 Met Glu Met Phe Thr
Phe Leu Leu Thr Cys Ile Phe Leu Pro Leu Leu 1 5 10 15 Arg Gly His
Ser Leu Phe Thr Cys Glu Pro Ile Thr Val Pro Arg Cys 20 25 30 Met
Lys Met Ala Tyr Asn Met Thr Phe Phe Pro Asn Leu Met Gly His 35 40
45 Tyr Asp Gln Ser Ile Ala Ala Val Glu Met Glu His Phe Leu Pro Leu
50 55 60 Ala Asn Leu Glu Cys Ser Pro Asn Ile Glu Thr Phe Leu Cys
Lys Ala 65 70 75 80 Phe Val Pro Thr Cys Ile Glu Gln Ile His Val Val
Pro Pro Cys Arg 85 90 95 Lys Leu Cys Glu Lys Val Tyr Ser Asp Cys
Lys Lys Leu Ile Asp Thr 100 105 110 Phe Gly Ile Arg Trp Pro Glu Glu
Leu Glu Cys Asp Arg Leu Gln Tyr 115 120 125 Cys Asp Glu Thr Val Pro
Val Thr Phe Asp Pro His Thr Glu Phe Leu 130 135 140 Gly Pro Gln Lys
Lys Thr Glu Gln Val Gln Arg Asp Ile Gly Phe Trp 145 150 155 160 Cys
Pro Arg His Leu Lys Thr Ser Gly Gly Gln Gly Tyr Lys Phe Leu 165 170
175 Gly Ile Asp Gln Cys Ala Pro Pro Cys Pro Asn Met Tyr Phe Lys Ser
180 185 190 Asp Glu Leu Glu Phe Ala Lys Ser Phe Ile Gly Thr Val Ser
Ile Phe 195 200 205 Cys Leu Cys Ala Thr Leu Phe Thr Phe Leu Thr Phe
Leu Ile Asp Val 210 215 220 Arg Arg Phe Arg Tyr Pro Glu Arg Pro Ile
Ile Tyr Tyr Ser Val Cys 225 230 235 240 Tyr Ser Ile Val Ser Leu Met
Tyr Phe Ile Gly Phe Leu Leu Gly Asp 245 250 255 Ser Thr Ala Cys Asn
Lys Ala Asp Glu Lys Leu Glu Leu Gly Asp Thr 260 265 270 Val Val Leu
Gly Ser Gln Asn Lys Ala Cys Thr Val Leu Phe Met Leu 275 280 285 Leu
Tyr Phe Phe Thr Met Ala Gly Thr Val Trp Trp Val Ile Leu Thr 290 295
300 Ile Thr Trp Phe Leu Ala Ala Gly Arg Lys Trp Ser Cys Glu Ala Ile
305 310 315 320 Glu Gln Lys Ala Val Trp Phe His Ala Val Ala Trp Gly
Thr Pro Gly 325 330 335 Phe Leu Thr Val Met Leu Leu Ala Leu Asn Lys
Val Glu Gly Asp Asn 340 345 350 Ile Ser Gly Val Cys Phe Val Gly Leu
Tyr Asp Leu Asp Ala Ser Arg 355 360 365 Tyr Phe Val Leu Leu Pro Leu
Cys Leu Cys Val Phe Val Gly Leu Ser 370 375 380 Leu Leu Leu Ala Gly
Ile Ile Ser Leu Asn His Val Arg Gln Val Ile 385 390 395 400 Gln His
Asp Gly Arg Asn Gln Glu Lys Leu Lys Lys Phe Met Ile Arg 405 410 415
Ile Gly Val Phe Ser Gly Leu Tyr Leu Val Pro Leu Val Thr Leu Leu 420
425 430 Gly Cys Tyr Val Tyr Glu Gln Val Asn Arg Ile Thr Trp Glu Ile
Thr 435 440 445 Trp Val Ser Asp His Cys Arg Gln Tyr His Ile Pro Cys
Pro Tyr Gln 450 455 460 Ala Lys Ala Lys Ala Arg Pro Glu Leu Ala Leu
Phe Met Ile Lys Tyr 465 470 475 480 Leu Met Thr Leu Ile Val Gly Ile
Ser Ala Val Phe Trp Val Gly Ser 485 490 495 Lys Lys Thr Cys Thr Glu
Trp Ala Gly Phe Phe Lys Arg Asn Arg Lys 500 505 510 Arg Asp Pro Ile
Ser Glu Ser Arg Arg Val Leu Gln Glu Ser Cys Glu 515 520 525 Phe Phe
Leu Lys His Asn Ser Lys Val Lys His Lys Lys Lys His Tyr 530 535 540
Lys Pro Ser Ser His Lys Leu Lys Val Ile Ser Lys Ser Met Gly Thr 545
550 555 560 Ser Thr Gly Ala Thr Ala Asn His Gly Thr Ser Ala Val Ala
Ile Thr 565 570 575 Ser His Asp Tyr Leu Gly Gln Glu Thr Leu Thr Glu
Ile Gln Thr Ser 580 585 590 Pro Glu Thr Ser Met Arg Glu Val Lys Ala
Asp Gly Ala Ser Thr Pro 595 600 605 Arg Leu Arg Glu Gln Asp Cys Gly
Glu Pro Ala Ser Pro Ala Ala Ser 610 615 620 Ile Ser Arg Leu Ser Gly
Glu Gln Val Asp Gly Lys Gly Gln Ala Gly 625 630 635 640 Ser Val Ser
Glu Ser Ala Arg Ser Glu Gly Arg Ile Ser Pro Lys Ser 645 650 655 Asp
Ile Thr Asp Thr Gly Leu Ala Gln Ser Asn Asn Leu Gln Val Pro 660 665
670 Ser Ser Ser Glu Pro Ser Ser Leu Lys Gly Ser Thr Ser Leu Leu Val
675 680 685 His Pro Val Ser Gly Val Arg Lys Glu Gln Gly Gly Gly Cys
His Ser 690 695 700 Asp Thr 705 50 3342 DNA Homo sapiens human
frizzled6 (Fzd6) 50 gcagctccag tcccggacgc aaccccggag ccgtctcagg
tccctggggg gaacggtggg 60 ttagacgggg acgggaaggg acagcggcct
tcgaccgccc cccgagtaat tgacccagga 120 ctcattttca ggaaagcctg
aaaatgagta aaatagtgaa atgaggaatt tgaacatttt 180 atctttggat
ggggatcttc tgaggatgca aagagtgatt catccaagcc atgtggtaaa 240
atcaggaatt tgaagaaaat ggagatgttt acatttttgt tgacgtgtat ttttctaccc
300 ctcctaagag ggcacagtct cttcacctgt gaaccaatta ctgttcccag
atgtatgaaa 360 atggcctaca acatgacgtt tttccctaat ctgatgggtc
attatgacca gagtattgcc 420 gcggtggaaa tggagcattt tcttcctctc
gcaaatctgg aatgttcacc aaacattgaa 480 actttcctct gcaaagcatt
tgtaccaacc tgcatagaac aaattcatgt ggttccacct 540 tgtcgtaaac
tttgtgagaa agtatattct gattgcaaaa aattaattga cacttttggg 600
atccgatggc ctgaggagct tgaatgtgac agattacaat actgtgatga gactgttcct
660 gtaacttttg atccacacac agaatttctt ggtcctcaga agaaaacaga
acaagtccaa 720 agagacattg gattttggtg tccaaggcat cttaagactt
ctgggggaca aggatataag 780 tttctgggaa ttgaccagtg tgcgcctcca
tgccccaaca tgtattttaa aagtgatgag 840 ctagagtttg caaaaagttt
tattggaaca gtttcaatat tttgtctttg tgcaactctg 900 ttcacattcc
ttactttttt aattgatgtt agaagattca gatacccaga gagaccaatt 960
atatattact ctgtctgtta cagcattgta tctcttatgt acttcattgg atttttgctg
1020 ggcgatagca cagcctgcaa taaggcagat gagaagctag aacttggtga
cactgttgtc 1080 ctaggctctc aaaataaggc ttgcaccgtt ttgttcatgc
ttttgtattt tttcacaatg 1140 gctggcactg tgtggtgggt gattcttacc
attacttggt tcttagctgc aggaagaaaa 1200 tggagttgtg aagccatcga
gcaaaaagca gtgtggtttc atgctgttgc atggggaaca 1260 ccaggtttcc
tgactgttat gcttcttgct ctgaacaaag ttgaaggaga caacattagt 1320
ggagtttgct ttgttggcct ttatgacctg gatgcttctc gctactttgt actcttgcca
1380 ctgtgccttt gtgtgtttgt tgggctctct cttcttttag ctggcattat
ttccttaaat 1440 catgttcgac aagtcataca acatgatggc cggaaccaag
aaaaactaaa gaaatttatg 1500 attcgaattg gagtcttcag cggcttgtat
cttgtgccat tagtgacact tctcggatgt 1560 tacgtctatg agcaagtgaa
caggattacc tgggagataa cttgggtctc tgatcattgt 1620 cgtcagtacc
atatcccatg tccttatcag gcaaaagcaa aagctcgacc agaattggct 1680
ttatttatga taaaatacct gatgacatta attgttggca tctctgctgt cttctgggtt
1740 ggaagcaaaa agacatgcac agaatgggct gggtttttta aacgaaatcg
caagagagat 1800 ccaatcagtg aaagtcgaag agtactacag gaatcatgtg
agtttttctt aaagcacaat 1860 tctaaagtta aacacaaaaa gaagcactat
aaaccaagtt cacacaagct gaaggtcatt 1920 tccaaatcca tgggaaccag
cacaggagct acagcaaatc atggcacttc tgcagtagca 1980 attactagcc
atgattacct aggacaagaa actttgacag aaatccaaac ctcaccagaa 2040
acatcaatga gagaggtgaa agcggacgga gctagcaccc ccaggttaag agaacaggac
2100 tgtggtgaac ctgcctcgcc agcagcatcc atctccagac tctctgggga
acaggtcgac 2160 gggaagggcc aggcaggcag tgtatctgaa agtgcgcgga
gtgaaggaag gattagtcca 2220 aagagtgata ttactgacac tggcctggca
cagagcaaca atttgcaggt ccccagttct 2280 tcagaaccaa gcagcctcaa
aggttccaca tctctgcttg ttcacccagt ttcaggagtg 2340 agaaaagagc
agggaggtgg ttgtcattca gatacttgaa gaacattttc tctcgttact 2400
cagaagcaaa tttgtgttac actggaagtg acctatgcac tgttttgtaa gaatcactgt
2460 tacgttcttc ttttgcactt aaagttgcat tgcctactgt tatactggaa
aaaatagagt 2520 tcaagaataa tatgactcat ttcacacaaa ggttaatgac
aacaatatac ctgaaaacag 2580 aaatgtgcag gttaataata tttttttaat
agtgtgggag gacagagtta gaggaatctt 2640 ccttttctat ttatgaagat
tctactcttg gtaagagtat tttaagatgt actatgctat 2700 tttacctttt
tgatataaaa tcaagatatt tctttgctga agtatttaaa tcttatcctt 2760
gtatcttttt atacatattt gaaaataagc ttatatgtat ttgaactttt ttgaaatcct
2820 attcaagtat ttttatcatg ctattgtgat attttagcac tttggtagct
tttacactga 2880 atttctaaga aaattgtaaa atagtcttct tttatactgt
aaaaaaagat ataccaaaaa 2940 gtcttataat aggaatttaa ctttaaaaac
ccacttattg ataccttacc atctaaaatg 3000 tgtgattttt atagtctcgt
tttaggaatt tcacagatct aaattatgta actgaaataa 3060 ggtgcttact
caaagagtgt ccactattga ttgtattatg ctgctcactg atccttctgc 3120
atatttaaaa taaaatgtcc taaagggtta gtagacaaaa tgttagtctt ttgtatatta
3180 ggccaagtgc aattgacttc ccttttttaa tgtttcatga ccacccattg
attgtattat 3240 aaccacttac agttgcttat attttttgtt ttaacttttg
tttcttaaca tttagaatat 3300 tacattttgt attatacagt acctttctca
gacattttgt ag 3342 51 574 PRT Homo sapiens human frizzled7 (Fzd7)
51 Met Arg Asp Pro Gly Ala Ala Ala Pro Leu Ser Ser Leu Gly Leu Cys
1 5 10 15 Ala Leu Val Leu Ala Leu Leu Gly Ala Leu Ser Ala Gly Ala
Gly Ala 20 25 30 Gln Pro Tyr His Gly Glu Lys Gly Ile Ser Val Pro
Asp His Gly Phe 35 40 45 Cys Gln Pro Ile Ser Ile Pro Leu Cys Thr
Asp Ile Ala Tyr Asn Gln 50 55 60 Thr Ile Leu Pro Asn Leu Leu Gly
His Thr Asn Gln Glu Asp Ala Gly 65 70 75 80 Leu Glu Val His Gln Phe
Tyr Pro Leu Val Lys Val Gln Cys Ser Pro 85 90 95 Glu Leu Arg Phe
Phe Leu Cys Ser Met Tyr Ala Pro Val Cys Thr Val 100 105 110 Leu Asp
Gln Ala Ile Pro Pro Cys Arg Ser Leu Cys Glu Arg Ala Arg 115 120 125
Gln Gly Cys Glu Ala Leu Met Asn Lys Phe Gly Phe Gln Trp Pro Glu 130
135 140 Arg Leu Arg Cys Glu Asn Phe Pro Val His Gly Ala Gly Glu Ile
Cys 145 150 155 160 Val Gly Gln Asn Thr Ser Asp Gly Ser Gly Gly Pro
Gly Gly Gly Pro 165 170 175 Thr Ala Tyr Pro Thr Ala Pro Tyr Leu Pro
Asp Leu Pro Phe Thr Ala 180 185 190 Leu Pro Pro Gly Ala Ser Asp Gly
Arg Gly Arg Pro Ala Phe Pro Phe 195 200 205 Ser Cys Pro Arg Gln Leu
Lys Val Pro Pro Tyr Leu Gly Tyr Arg Phe 210 215 220 Leu Gly Glu Arg
Asp Cys Gly Ala Pro Cys Glu Pro Gly Arg Ala Asn 225 230 235 240 Gly
Leu Met Tyr Phe Lys Glu Glu Glu Arg Arg Phe Ala Arg Leu Trp 245 250
255 Val Gly Val Trp Ser Val Leu Cys Cys Ala Ser Thr Leu Phe Thr Val
260 265 270 Leu Thr Tyr Leu Val Asp Met Arg Arg Phe Ser Tyr Pro Glu
Arg Pro 275 280 285 Ile Ile Phe Leu Ser Gly Cys Tyr Phe Met Val Ala
Val Ala His Val 290 295 300 Ala Gly Phe Leu Leu Glu Asp Arg Ala Val
Cys Val Glu Arg Phe Ser 305 310 315 320 Asp Asp Gly Tyr Arg Thr Val
Ala Gln Gly Thr Lys Lys Glu Gly Cys 325 330 335 Thr Ile Leu Phe Met
Val Leu Tyr Phe Phe Gly Met Ala Ser Ser Ile 340 345 350 Trp Trp Val
Ile Leu Ser Leu Thr Trp Phe Leu Ala Ala Gly Met Lys 355 360 365 Trp
Gly His Glu Ala Ile Glu Ala Asn Ser Gln Tyr Phe His Leu Ala 370 375
380 Ala Trp Ala Val Pro Ala Val Lys Thr Ile Thr Ile Leu Ala Met Gly
385 390 395 400 Gln Val Asp Gly Asp Leu Leu Ser Gly Val Cys Tyr Val
Gly Leu Ser 405 410 415 Ser Val Asp Ala Leu Arg Gly Phe Val Leu Ala
Pro Leu Phe Val Tyr 420 425 430 Leu Phe Ile Gly Thr Ser Phe Leu Leu
Ala Gly Phe Val Ser Leu Phe 435 440 445 Arg Ile Arg Thr Ile Met Lys
His Asp Gly Thr Lys Thr Glu Lys Leu 450 455 460 Glu Lys Leu Met Val
Arg Ile Gly Val Phe Ser Val Leu Tyr Thr Val 465 470 475 480 Pro Ala
Thr Ile Val Leu Ala Cys Tyr Phe Tyr Glu Gln Ala Phe Arg 485 490 495
Glu His Trp Glu Arg Thr Trp Leu Leu Gln Thr Cys Lys Ser Tyr Ala 500
505 510 Val Pro Cys Pro Pro Gly His Phe Pro Pro Met Ser Pro Asp Phe
Thr 515 520 525 Val Phe Met Ile Lys Tyr Leu Met Thr Met Ile Val Gly
Ile Thr Thr 530 535 540 Gly Phe Trp Ile Trp Ser Gly Lys Thr Leu Gln
Ser Trp Arg Arg Phe 545 550 555 560 Tyr His
Arg Leu Ser His Ser Ser Lys Gly Glu Thr Ala Val 565 570 52 3851 DNA
Homo sapiens human frizzled7 (Fzd7) 52 ctctcccaac cgcctcgtcg
cactcctcag gctgagagca ccgctgcact cgcggccggc 60 gatgcgggac
cccggcgcgg ccgctccgct ttcgtccctg ggcctctgtg ccctggtgct 120
ggcgctgctg ggcgcactgt ccgcgggcgc cggggcgcag ccgtaccacg gagagaaggg
180 catctccgtg ccggaccacg gcttctgcca gcccatctcc atcccgctgt
gcacggacat 240 cgcctacaac cagaccatcc tgcccaacct gctgggccac
acgaaccaag aggacgcggg 300 cctcgaggtg caccagttct acccgctggt
gaaggtgcag tgttctcccg aactccgctt 360 tttcttatgc tccatgtatg
cgcccgtgtg caccgtgctc gatcaggcca tcccgccgtg 420 tcgttctctg
tgcgagcgcg cccgccaggg ctgcgaggcg ctcatgaaca agttcggctt 480
ccagtggccc gagcggctgc gctgcgagaa cttcccggtg cacggtgcgg gcgagatctg
540 cgtgggccag aacacgtcgg acggctccgg gggcccaggc ggcggcccca
ctgcctaccc 600 taccgcgccc tacctgccgg acctgccctt caccgcgctg
cccccggggg cctcagatgg 660 cagggggcgt cccgccttcc ccttctcatg
cccccgtcag ctcaaggtgc ccccgtacct 720 gggctaccgc ttcctgggtg
agcgcgattg tggcgccccg tgcgaaccgg gccgtgccaa 780 cggcctgatg
tactttaagg aggaggagag gcgcttcgcc cgcctctggg tgggcgtgtg 840
gtccgtgctg tgctgcgcct cgacgctctt taccgttctc acctacctgg tggacatgcg
900 gcgcttcagc tacccagagc ggcccatcat cttcctgtcg ggctgctact
tcatggtggc 960 cgtggcgcac gtggccggct tccttctaga ggaccgcgcc
gtgtgcgtgg agcgcttctc 1020 ggacgatggc taccgcacgg tggcgcaggg
caccaagaag gagggctgca ccatcctctt 1080 catggtgctc tacttcttcg
gcatggccag ctccatctgg tgggtcattc tgtctctcac 1140 ttggttcctg
gcggccggca tgaagtgggg ccacgaggcc atcgaggcca actcgcagta 1200
cttccacctg gccgcgtggg ccgtgcccgc cgtcaagacc atcactatcc tggccatggg
1260 ccaggtagac ggggacctgc tgagcggggt gtgctacgtt ggcctctcca
gtgtggacgc 1320 gctgcggggc ttcgtgctgg cgcctctgtt cgtctacctc
ttcataggca cgtccttctt 1380 gctggccggc ttcgtgtccc tcttccgtat
ccgcaccatc atgaaacacg acggcaccaa 1440 gaccgagaag ctggagaagc
tcatggtgcg catcggcgtc ttcagcgtgc tctacacagt 1500 gcccgccacc
atcgtcctgg cctgctactt ctacgagcag gccttccgcg agcactggga 1560
gcgcacctgg ctcctgcaga cgtgcaagag ctatgccgtg ccctgcccgc ccggccactt
1620 cccgcccatg agccccgact tcaccgtctt catgatcaag tacctgatga
ccatgatcgt 1680 cggcatcacc actggcttct ggatctggtc gggcaagacc
ctgcagtcgt ggcgccgctt 1740 ctaccacaga cttagccaca gcagcaaggg
ggagactgcg gtatgagccc cggcccctcc 1800 ccacctttcc caccccagcc
ctcttgcaag aggagaggca cggtagggaa aagaactgct 1860 gggtgggggc
ctgtttctgt aactttctcc ccctctactg agaagtgacc tggaagtgag 1920
aagttctttg cagatttggg gcgaggggtg atttggaaaa gaagacctgg gtggaaagcg
1980 gtttggatga aaagatttca ggcaaagact tgcaggaaga tgatgataac
ggcgatgtga 2040 atcgtcaaag gtacgggcca gcttgtgcct aatagaaggt
tgagaccagc agagactgct 2100 gtgagtttct cccggctccg aggctgaacg
gggactgtga gcgatccccc tgctgcaggg 2160 cgagtggcct gtccagaccc
ctgtgaggcc ccgggaaagg tacagccctg tctgcggtgg 2220 ctgctttgtt
ggaaagaggg agggcctcct gcggtgtgct tgtcaagcag tggtcaaacc 2280
ataatctctt ttcactgggg ccaaactgga gcccagatgg gttaatttcc agggtcagac
2340 attacggtct ctcctcccct gccccctccc gcctgttttt cctcccgtac
tgctttcagg 2400 tcttgtaaaa taagcatttg gaagtcttgg gaggcctgcc
tgctagaatc ctaatgtgag 2460 gatgcaaaag aaatgatgat aacattttga
gataaggcca aggagacgtg gagtaggtat 2520 ttttgctact ttttcatttt
ctggggaagg caggaggcag aaagacgggt gttttatttg 2580 gtctaatacc
ctgaaaagaa gtgatgactt gttgcttttc aaaacaggaa tgcatttttc 2640
cccttgtctt tgttgtaaga gacaaaagag gaaacaaaag tgtctccctg tggaaaggca
2700 taactgtgac gaaagcaact tttataggca aagcagcgca aatctgaggt
ttcccgttgg 2760 ttgttaattt ggttgagata aacattcctt tttaaggaaa
agtgaagagc agtgtgctgt 2820 cacacaccgt taagccagag gttctgactt
cgctaaagga aatgtaagag gttttgttgt 2880 ctgttttaaa taaatttaat
tcggaacaca tgatccaaca gactatgtta aaatattcag 2940 ggaaatctct
cccttcattt actttttctt gctataagcc tatatttagg tttcttttct 3000
atttttttct cccatttgga tcctttgagg taaaaaaaca taatgtcttc agcctcataa
3060 taaaggaaag ttaattaaaa aaaaaaagca aagagccatt ttgtcctgtt
ttcttggttc 3120 catcaatctg tttattaaac atcatccata tgctgaccct
gtctctgtgt ggttgggttg 3180 ggaggcgatc agcagatacc atagtgaacg
aagaggaagg tttgaaccat gggccccatc 3240 tttaaagaaa gtcattaaaa
gaaggtaaac ttcaaagtga ttctggagtt ctttgaaatg 3300 tgctggaaga
cttaaattta ttaatcttaa atcatgtact ttttttctgt aatagaactc 3360
ggattctttt gcatgatggg gtaaagctta gcagagaatc atgggagcta acctttatcc
3420 cacctttgac actaccctcc aatcttgcaa cactatcctg tttctcagaa
cagtttttaa 3480 atgccaatca tagagggtac tgtaaagtgt acaagttact
ttatatatgt aatgttcact 3540 tgagtggaac tgctttttac attaaagtta
aaatcgatct tgtgtttctt caaccttcaa 3600 aactatctca tctgtcagat
ttttaaaact ccaacacagg ttttggcatc ttttgtgctg 3660 tatcttttaa
gtgcatgtga aatttgtaaa atagagataa gtacagtatg tatattttgt 3720
aaatctccca tttttgtaag aaaatatata ttgtatttat acatttttac tttggatttt
3780 tgttttgttg gctttaaagg tctaccccac tttatcacat gtacagatca
caaataaatt 3840 tttttaaata c 3851 53 694 PRT Homo sapiens human
frizzled8 (Fzd8) 53 Met Glu Trp Gly Tyr Leu Leu Glu Val Thr Ser Leu
Leu Ala Ala Leu 1 5 10 15 Ala Leu Leu Gln Arg Ser Ser Gly Ala Ala
Ala Ala Ser Ala Lys Glu 20 25 30 Leu Ala Cys Gln Glu Ile Thr Val
Pro Leu Cys Lys Gly Ile Gly Tyr 35 40 45 Asn Tyr Thr Tyr Met Pro
Asn Gln Phe Asn His Asp Thr Gln Asp Glu 50 55 60 Ala Gly Leu Glu
Val His Gln Phe Trp Pro Leu Val Glu Ile Gln Cys 65 70 75 80 Ser Pro
Asp Leu Lys Phe Phe Leu Cys Ser Met Tyr Thr Pro Ile Cys 85 90 95
Leu Glu Asp Tyr Lys Lys Pro Leu Pro Pro Cys Arg Ser Val Cys Glu 100
105 110 Arg Ala Lys Ala Gly Cys Ala Pro Leu Met Arg Gln Tyr Gly Phe
Ala 115 120 125 Trp Pro Asp Arg Met Arg Cys Asp Arg Leu Pro Glu Gln
Gly Asn Pro 130 135 140 Asp Thr Leu Cys Met Asp Tyr Asn Arg Thr Asp
Leu Thr Thr Ala Ala 145 150 155 160 Pro Ser Pro Pro Arg Arg Leu Pro
Pro Pro Pro Pro Gly Glu Gln Pro 165 170 175 Pro Ser Gly Ser Gly His
Gly Arg Pro Pro Gly Ala Arg Pro Pro His 180 185 190 Arg Gly Gly Gly
Arg Gly Gly Gly Gly Gly Asp Ala Ala Ala Pro Pro 195 200 205 Ala Arg
Gly Gly Gly Gly Gly Gly Lys Ala Arg Pro Pro Gly Gly Gly 210 215 220
Ala Ala Pro Cys Glu Pro Gly Cys Gln Cys Arg Ala Pro Met Val Ser 225
230 235 240 Val Ser Ser Glu Arg His Pro Leu Tyr Asn Arg Val Lys Thr
Gly Gln 245 250 255 Ile Ala Asn Cys Ala Leu Pro Cys His Asn Pro Phe
Phe Ser Gln Asp 260 265 270 Glu Arg Ala Phe Thr Val Phe Trp Ile Gly
Leu Trp Ser Val Leu Cys 275 280 285 Phe Val Ser Thr Phe Ala Thr Val
Ser Thr Phe Leu Ile Asp Met Glu 290 295 300 Arg Phe Lys Tyr Pro Glu
Arg Pro Ile Ile Phe Leu Ser Ala Cys Tyr 305 310 315 320 Leu Phe Val
Ser Val Gly Tyr Leu Val Arg Leu Val Ala Gly His Glu 325 330 335 Lys
Val Ala Cys Ser Gly Gly Ala Pro Gly Ala Gly Gly Ala Gly Gly 340 345
350 Ala Gly Gly Ala Ala Ala Gly Ala Gly Ala Ala Gly Ala Gly Ala Gly
355 360 365 Gly Pro Gly Gly Arg Gly Glu Tyr Glu Glu Leu Gly Ala Val
Glu Gln 370 375 380 His Val Arg Tyr Glu Thr Thr Gly Pro Ala Leu Cys
Thr Val Val Phe 385 390 395 400 Leu Leu Val Tyr Phe Phe Gly Met Ala
Ser Ser Ile Trp Trp Val Ile 405 410 415 Leu Ser Leu Thr Trp Phe Leu
Ala Ala Gly Met Lys Trp Gly Asn Glu 420 425 430 Ala Ile Ala Gly Tyr
Ser Gln Tyr Phe His Leu Ala Ala Trp Leu Val 435 440 445 Pro Ser Val
Lys Ser Ile Ala Val Leu Ala Leu Ser Ser Val Asp Gly 450 455 460 Asp
Pro Val Ala Gly Ile Cys Tyr Val Gly Asn Gln Ser Leu Asp Asn 465 470
475 480 Leu Arg Gly Phe Val Leu Ala Pro Leu Val Ile Tyr Leu Phe Ile
Gly 485 490 495 Thr Met Phe Leu Leu Ala Gly Phe Val Ser Leu Phe Arg
Ile Arg Ser 500 505 510 Val Ile Lys Gln Gln Asp Gly Pro Thr Lys Thr
His Lys Leu Glu Lys 515 520 525 Leu Met Ile Arg Leu Gly Leu Phe Thr
Val Leu Tyr Thr Val Pro Ala 530 535 540 Ala Val Val Val Ala Cys Leu
Phe Tyr Glu Gln His Asn Arg Pro Arg 545 550 555 560 Trp Glu Ala Thr
His Asn Cys Pro Cys Leu Arg Asp Leu Gln Pro Asp 565 570 575 Gln Ala
Arg Arg Pro Asp Tyr Ala Val Phe Met Leu Lys Tyr Phe Met 580 585 590
Cys Leu Val Val Gly Ile Thr Ser Gly Val Trp Val Trp Ser Gly Lys 595
600 605 Thr Leu Glu Ser Trp Arg Ser Leu Cys Thr Arg Cys Cys Trp Ala
Ser 610 615 620 Lys Gly Ala Ala Val Gly Gly Gly Ala Gly Ala Thr Ala
Ala Gly Gly 625 630 635 640 Gly Gly Gly Pro Gly Gly Gly Gly Gly Gly
Gly Pro Gly Gly Gly Gly 645 650 655 Gly Pro Gly Gly Gly Gly Gly Ser
Leu Tyr Ser Asp Val Ser Thr Gly 660 665 670 Leu Thr Trp Arg Ser Gly
Thr Ala Ser Ser Val Ser Tyr Pro Lys Gln 675 680 685 Met Pro Leu Ser
Gln Val 690 54 3195 DNA Homo sapiens human frizzled8 (Fzd8) 54
acagcatgga gtggggttac ctgttggaag tgacctcgct gctggccgcc ttggcgctgc
60 tgcagcgctc tagcggcgct gcggccgcct cggccaagga gctggcatgc
caagagatca 120 ccgtgccgct gtgtaagggc atcggctaca actacaccta
catgcccaat cagttcaacc 180 acgacacgca agacgaggcg ggcctggagg
tgcaccagtt ctggccgctg gtggagatcc 240 agtgctcgcc cgatctcaag
ttcttcctgt gcagcatgta cacgcccatc tgcctagagg 300 actacaagaa
gccgctgccg ccctgccgct cggtgtgcga gcgcgccaag gccggctgcg 360
cgccgctcat gcgccagtac ggcttcgcct ggcccgaccg catgcgctgc gaccggctgc
420 ccgagcaagg caaccctgac acgctgtgca tggactacaa ccgcaccgac
ctaaccaccg 480 ccgcgcccag cccgccgcgc cgcctgccgc cgccgccgcc
cggcgagcag ccgccttcgg 540 gcagcggcca cggccgcccg ccgggggcca
ggcccccgca ccgcggaggc ggcaggggcg 600 gtggcggcgg ggacgcggcg
gcgcccccag ctcgcggcgg cggcggtggc gggaaggcgc 660 ggccccctgg
cggcggcgcg gctccctgcg agcccgggtg ccagtgccgc gcgcctatgg 720
tgagcgtgtc cagcgagcgc cacccgctct acaaccgcgt caagacaggc cagatcgcta
780 actgcgcgct gccctgccac aacccctttt tcagccagga cgagcgcgcc
ttcaccgtct 840 tctggatcgg cctgtggtcg gtgctctgct tcgtgtccac
cttcgccacc gtctccacct 900 tccttatcga catggagcgc ttcaagtacc
cggagcggcc cattatcttc ctctcggcct 960 gctacctctt cgtgtcggtg
ggctacctag tgcgcctggt ggcgggccac gagaaggtgg 1020 cgtgcagcgg
tggcgcgccg ggcgcggggg gcgctggggg cgcgggcggc gcggcggcgg 1080
gcgcgggcgc ggcgggcgcg ggcgcgggcg gcccgggcgg gcgcggcgag tacgaggagc
1140 tgggcgcggt ggagcagcac gtgcgctacg agaccaccgg ccccgcgctg
tgcaccgtgg 1200 tcttcttgct ggtctacttc ttcggcatgg ccagctccat
ctggtgggtg atcttgtcgc 1260 tcacatggtt cctggcggcc ggtatgaagt
ggggcaacga agccatcgcc ggctactcgc 1320 agtacttcca cctggccgcg
tggcttgtgc ccagcgtcaa gtccatcgcg gtgctggcgc 1380 tcagctcggt
ggacggcgac ccggtggcgg gcatctgcta cgtgggcaac cagagcctgg 1440
acaacctgcg cggcttcgtg ctggcgccgc tggtcatcta cctcttcatc ggcaccatgt
1500 tcctgctggc cggcttcgtg tccctgttcc gcatccgctc ggtcatcaag
caacaggacg 1560 gccccaccaa gacgcacaag ctggagaagc tgatgatccg
cctgggcctg ttcaccgtgc 1620 tctacaccgt gcccgccgcg gtggtggtcg
cctgcctctt ctacgagcag cacaaccgcc 1680 cgcgctggga ggccacgcac
aactgcccgt gcctgcggga cctgcagccc gaccaggcac 1740 gcaggcccga
ctacgccgtc ttcatgctca agtacttcat gtgcctagtg gtgggcatca 1800
cctcgggcgt gtgggtctgg tccggcaaga cgctggagtc ctggcgctcc ctgtgcaccc
1860 gctgctgctg ggccagcaag ggcgccgcgg tgggcggggg cgcgggcgcc
acggccgcgg 1920 ggggtggcgg cgggccgggg ggcggcggcg gcgggggacc
cggcggcggc ggggggccgg 1980 gcggcggcgg gggctccctc tacagcgacg
tcagcactgg cctgacgtgg cggtcgggca 2040 cggcgagctc cgtgtcttat
ccaaagcaga tgccattgtc ccaggtctga gcggagggga 2100 gggggcgccc
aggaggggtg gggagggggg cgaggagacc caagtgcagc gaagggacac 2160
ttgatgggct gaggttccca ccccttcaca gtgttgattg ctattagcat gataatgaac
2220 tcttaatggt atccattagc tgggacttaa atgactcact tagaacaaag
tacctggcat 2280 tgaagcctcc cagacccagc cccttttcct ccattgatgt
gcggggagct cctcccgcca 2340 cgcgttaatt tctgttggct gaggagggtg
gactctgcgg cgtttccaga acccgagatt 2400 tggagccctc cctggctgca
cttggctggg tttgcagtca gatacacaga tttcacctgg 2460 gagaacctct
ttttctccct cgactcttcc tacgtaaact cccacccctg acttaccctg 2520
gaggaggggt gaccgccacc tgatgggatt gcacggtttg ggtattctta atgaccaggc
2580 aaatgcctta agtaaacaaa caagaaatgt cttaattata caccccacgt
aaatacgggt 2640 ttcttacatt agaggatgta tttatataat tatttgttaa
attgtaaaaa aaaaaagtgt 2700 aaaatatgta tatatccaaa gatatagtgt
gtacattttt ttgtaaaaag tttagaggct 2760 tacccctgta agaacagata
taagtattct attttgtcaa taaaatgact tttgataaat 2820 gatttaacca
ttgccctctc ccccgcctct tctgagctgt cacctttaaa gtgcttgcta 2880
aggacgcatg gggaaaatgg acattttctg gcttgtcatt ctgtacactg accttaggca
2940 tggagaaaat tacttgttaa actctagttc ttaagttgtt agccaagtaa
atatcattgt 3000 tgaactgaaa tcaaaattga gtttttgcac cttccccaaa
gacggtgttt ttcatgggag 3060 ctcttttctg atccatggat aacaactctc
actttagtgg atgtaaatgg aacttctgca 3120 aggcagtaat tccccttagg
ccttgttatt tatcctgcat ggtatcacta aaggtttcaa 3180 aaccctgaaa aaaaa
3195 55 591 PRT Homo sapiens human frizzled9 (Fzd9) 55 Met Ala Val
Ala Pro Leu Arg Gly Ala Leu Leu Leu Trp Gln Leu Leu 1 5 10 15 la
Ala Gly Gly Ala Ala Leu Glu Ile Gly Arg Phe Asp Pro Glu Arg 20 25
30 ly Arg Gly Ala Ala Pro Cys Gln Ala Val Glu Ile Pro Met Cys Arg
35 40 45 ly Ile Gly Tyr Asn Leu Thr Arg Met Pro Asn Leu Leu Gly His
Thr 50 55 60 er Gln Gly Glu Ala Ala Ala Glu Leu Ala Glu Phe Ala Pro
Leu Val 65 70 75 80 ln Tyr Gly Cys His Ser His Leu Arg Phe Phe Leu
Cys Ser Leu Tyr 85 90 95 la Pro Met Cys Thr Asp Gln Val Ser Thr Pro
Ile Pro Ala Cys Arg 100 105 110 ro Met Cys Glu Gln Ala Arg Leu Arg
Cys Ala Pro Ile Met Glu Gln 115 120 125 he Asn Phe Gly Trp Pro Asp
Ser Leu Asp Cys Ala Arg Leu Pro Thr 130 135 140 rg Asn Asp Pro His
Ala Leu Cys Met Glu Ala Pro Glu Asn Ala Thr 145 150 155 160 la Gly
Pro Ala Glu Pro His Lys Gly Leu Gly Met Leu Pro Val Ala 165 170 175
ro Arg Pro Ala Arg Pro Pro Gly Asp Leu Gly Pro Gly Ala Gly Gly 180
185 190 er Gly Thr Cys Glu Asn Pro Glu Lys Phe Gln Tyr Val Glu Lys
Ser 195 200 205 rg Ser Cys Ala Pro Arg Cys Gly Pro Gly Val Glu Val
Phe Trp Ser 210 215 220 rg Arg Asp Lys Asp Phe Ala Leu Val Trp Met
Ala Val Trp Ser Ala 225 230 235 240 eu Cys Phe Phe Ser Thr Ala Phe
Thr Val Leu Thr Phe Leu Leu Glu 245 250 255 ro His Arg Phe Gln Tyr
Pro Glu Arg Pro Ile Ile Phe Leu Ser Met 260 265 270 ys Tyr Asn Val
Tyr Ser Leu Ala Phe Leu Ile Arg Ala Val Ala Gly 275 280 285 la Gln
Ser Val Ala Cys Asp Gln Glu Ala Gly Ala Leu Tyr Val Ile 290 295 300
ln Glu Gly Leu Glu Asn Thr Gly Cys Thr Leu Val Phe Leu Leu Leu 305
310 315 320 yr Tyr Phe Gly Met Ala Ser Ser Leu Trp Trp Val Val Leu
Thr Leu 325 330 335 hr Trp Phe Leu Ala Ala Gly Lys Lys Trp Gly His
Glu Ala Ile Glu 340 345 350 la His Gly Ser Tyr Phe His Met Ala Ala
Trp Gly Leu Pro Ala Leu 355 360 365 ys Thr Ile Val Ile Leu Thr Leu
Arg Lys Val Ala Gly Asp Glu Leu 370 375 380 hr Gly Leu Cys Tyr Val
Ala Ser Thr Asp Ala Ala Ala Leu Thr Gly 385 390 395 400 he Val Leu
Val Pro Leu Ser Gly Tyr Leu Val Leu Gly Ser Ser Phe 405 410 415 eu
Leu Thr Gly Phe Val Ala Leu Phe His Ile Arg Lys Ile Met Lys 420 425
430 hr Gly Gly Thr Asn Thr Glu Lys Leu Glu Lys Leu Met Val Lys Ile
435 440 445 ly Val Phe Ser Ile Leu Tyr Thr Val Pro Ala Thr Cys Val
Ile Val 450 455 460 ys Tyr Val Tyr Glu Arg Leu Asn Met Asp Phe Trp
Arg Leu Arg Ala 465 470 475 480 hr Glu Gln Pro Cys Ala Ala Ala Ala
Gly Pro Gly Gly Arg Arg Asp 485 490 495 ys Ser Leu Pro Gly Gly Ser
Val Pro Thr Val Ala Val Phe Met Leu 500 505 510 ys Ile Phe Met Ser
Leu Val Val Gly Ile Thr Ser Gly Val Trp Val 515 520 525 rp Ser Ser
Lys Thr Phe Gln Thr Trp Gln Ser Leu Cys Tyr Arg Lys 530 535 540 le
Ala Ala Gly Arg Ala Arg Ala Lys Ala Cys Arg Ala Pro Gly Ser 545
550
555 560 yr Gly Arg Gly Thr His Cys His Tyr Lys Ala Pro Thr Val Val
Leu 565 570 575 is Met Thr Lys Thr Asp Pro Ser Leu Glu Asn Pro Thr
His Leu 580 585 590 56 2184 DNA Homo sapiens human frizzled9 (Fzd9)
56 ccgccttcgg cccgggcctc ccgggatggc cgtggcgcct ctgcgggggg
cgctgctgct 60 gtggcagctg ctggcggcgg gcggcgcggc actggagatc
ggccgcttcg acccggagcg 120 cgggcgcggg gctgcgccgt gccaggcggt
ggagatcccc atgtgccgcg gcatcggcta 180 caacctgacc cgcatgccca
acctgctggg ccacacgtcg cagggcgagg cggctgccga 240 gctagcggag
ttcgcgccgc tggtgcagta cggctgccac agccacctgc gcttcttcct 300
gtgctcgctc tacgcgccca tgtgcaccga ccaggtctcg acgcccattc ccgcctgccg
360 gcccatgtgc gagcaggcgc gcctgcgctg cgcgcccatc atggagcagt
tcaacttcgg 420 ctggccggac tcgctcgact gcgcccggct gcccacgcgc
aacgacccgc acgcgctgtg 480 catggaggcg cccgagaacg ccacggccgg
ccccgcggag ccccacaagg gcctgggcat 540 gctgcccgtg gcgccgcggc
ccgcgcgccc tcccggagac ctgggcccgg gcgcgggcgg 600 cagtggcacc
tgcgagaacc ccgagaagtt ccagtacgtg gagaagagcc gctcgtgcgc 660
accgcgctgc gggcccggcg tcgaggtgtt ctggtcccgg cgcgacaagg acttcgcgct
720 ggtctggatg gccgtgtggt cggcgctgtg cttcttctcc accgccttca
ctgtgctcac 780 cttcttgctg gagccccacc gcttccagta ccccgagcgc
cccatcatct tcctctccat 840 gtgctacaac gtctactcgc tggccttcct
gatccgtgcg gtggccggag cgcagagcgt 900 ggcctgtgac caggaggcgg
gcgcgctcta cgtgatccag gagggcctgg agaacacggg 960 ctgcacgctg
gtcttcctac tgctctacta cttcggcatg gccagctcgc tctggtgggt 1020
ggtcctgacg ctcacctggt tcctggctgc cgggaagaaa tggggccacg aggccatcga
1080 ggcccacggc agctatttcc acatggctgc ctggggcctg cccgcgctca
agaccatcgt 1140 catcctgacc ctgcgcaagg tggcgggtga tgagctgact
gggctttgct acgtggccag 1200 cacggatgca gcagcgctca cgggcttcgt
gctggtgccc ctctctggct acctggtgct 1260 gggcagtagt ttcctcctga
ccggcttcgt ggccctcttc cacatccgca agatcatgaa 1320 gacgggcggc
accaacacag agaagctgga gaagctcatg gtcaagatcg gggtcttctc 1380
catcctctac acggtgcccg ccacctgcgt catcgtttgc tatgtctacg aacgcctcaa
1440 catggacttc tggcgccttc gggccacaga gcagccatgc gcagcggccg
cggggcccgg 1500 aggccggagg gactgctcgc tgccaggggg ctcggtgccc
accgtggcgg tcttcatgct 1560 caaaattttc atgtcactgg tggtggggat
caccagcggc gtctgggtgt ggagctccaa 1620 gactttccag acctggcaga
gcctgtgcta ccgcaagata gcagctggcc gggcccgggc 1680 caaggcctgc
cgcgcccccg ggagctacgg acgtggcacg cactgccact ataaggctcc 1740
caccgtggtc ttgcacatga ctaagacgga cccctctttg gagaacccca cacacctcta
1800 gccacacagg cctggcgcgg ggtggctgct gccccctcct tgccctccac
gccctgcccc 1860 ctgcatcccc tagagacagc tgactagcag ctgcccagct
gtcaaggtca ggcaagtgag 1920 caccggggac tgaggatcag ggcgggaccc
cgtgaggctc attaggggag atgggggtct 1980 cccctaatgc gggggctgga
ccaggctgag tccccacagg gtcctagtgg aggatgtgga 2040 ggggcggggc
agaggggtcc agccggagtt tatttaatga tgtaatttat tgttgcgttc 2100
ctctggaagc tgtgactgga ataaaccccc gcgtggcact gctgatcctc tctggctggg
2160 aagggggaag gtaggaggtg aggc 2184 57 581 PRT Homo sapiens human
frizzled10 (Fzd10) 57 Met Gln Arg Pro Gly Pro Arg Leu Trp Leu Val
Leu Gln Val Met Gly 1 5 10 15 Ser Cys Ala Ala Ile Ser Ser Met Asp
Met Glu Arg Pro Gly Asp Gly 20 25 30 Lys Cys Gln Pro Ile Glu Ile
Pro Met Cys Lys Asp Ile Gly Tyr Asn 35 40 45 Met Thr Arg Met Pro
Asn Leu Met Gly His Glu Asn Gln Arg Glu Ala 50 55 60 Ala Ile Gln
Leu His Glu Phe Ala Pro Leu Val Glu Tyr Gly Cys His 65 70 75 80 Gly
His Leu Arg Phe Phe Leu Cys Ser Leu Tyr Ala Pro Met Cys Thr 85 90
95 Glu Gln Val Ser Thr Pro Ile Pro Ala Cys Arg Val Met Cys Glu Gln
100 105 110 Ala Arg Leu Lys Cys Ser Pro Ile Met Glu Gln Phe Asn Phe
Lys Trp 115 120 125 Pro Asp Ser Leu Asp Cys Arg Lys Leu Pro Asn Lys
Asn Asp Pro Asn 130 135 140 Tyr Leu Cys Met Glu Ala Pro Asn Asn Gly
Ser Asp Glu Pro Thr Arg 145 150 155 160 Gly Ser Gly Leu Phe Pro Pro
Leu Phe Arg Pro Gln Arg Pro His Ser 165 170 175 Ala Gln Glu His Pro
Leu Lys Asp Gly Gly Pro Gly Arg Gly Gly Cys 180 185 190 Asp Asn Pro
Gly Lys Phe His His Val Glu Lys Ser Ala Ser Cys Ala 195 200 205 Pro
Leu Cys Thr Pro Gly Val Asp Val Tyr Trp Ser Arg Glu Asp Lys 210 215
220 Arg Phe Ala Val Val Trp Leu Ala Ile Trp Ala Val Leu Cys Phe Phe
225 230 235 240 Ser Ser Ala Phe Thr Val Leu Thr Phe Leu Ile Asp Pro
Ala Arg Phe 245 250 255 Arg Tyr Pro Glu Arg Pro Ile Ile Phe Leu Ser
Met Cys Tyr Cys Val 260 265 270 Tyr Ser Val Gly Tyr Leu Ile Arg Leu
Phe Ala Gly Ala Glu Ser Ile 275 280 285 Ala Cys Asp Arg Asp Ser Gly
Gln Leu Tyr Val Ile Gln Glu Gly Leu 290 295 300 Glu Ser Thr Gly Cys
Thr Leu Val Phe Leu Val Leu Tyr Tyr Phe Gly 305 310 315 320 Met Ala
Ser Ser Leu Trp Trp Val Val Leu Thr Leu Thr Trp Phe Leu 325 330 335
Ala Ala Gly Lys Lys Trp Gly His Glu Ala Ile Glu Ala Asn Ser Ser 340
345 350 Tyr Phe His Leu Ala Ala Trp Ala Ile Pro Ala Val Lys Thr Ile
Leu 355 360 365 Ile Leu Val Met Arg Arg Val Ala Gly Asp Glu Leu Thr
Gly Val Cys 370 375 380 Tyr Val Gly Ser Met Asp Val Asn Ala Leu Thr
Gly Phe Val Leu Ile 385 390 395 400 Pro Leu Ala Cys Tyr Leu Val Ile
Gly Thr Ser Phe Ile Leu Ser Gly 405 410 415 Phe Val Ala Leu Phe His
Ile Arg Arg Val Met Lys Thr Gly Gly Glu 420 425 430 Asn Thr Asp Lys
Leu Glu Lys Leu Met Val Arg Ile Gly Leu Phe Ser 435 440 445 Val Leu
Tyr Thr Val Pro Ala Thr Cys Val Ile Ala Cys Tyr Phe Tyr 450 455 460
Glu Arg Leu Asn Met Asp Tyr Trp Lys Ile Leu Ala Ala Gln His Lys 465
470 475 480 Cys Lys Met Asn Asn Gln Thr Lys Thr Leu Asp Cys Leu Met
Ala Ala 485 490 495 Ser Ile Pro Ala Val Glu Ile Phe Met Val Lys Ile
Phe Met Leu Leu 500 505 510 Val Val Gly Ile Thr Ser Gly Met Trp Ile
Trp Thr Ser Lys Thr Leu 515 520 525 Gln Ser Trp Gln Gln Val Cys Ser
Arg Arg Leu Lys Lys Lys Ser Arg 530 535 540 Arg Lys Pro Ala Ser Val
Ile Thr Ser Gly Gly Ile Tyr Lys Lys Ala 545 550 555 560 Gln His Pro
Gln Lys Thr His His Gly Lys Tyr Glu Ile Pro Ala Gln 565 570 575 Ser
Pro Thr Cys Val 580 58 3260 DNA Homo sapiens human frizzled10
(Fzd10) 58 tcgaaacagc tgccggctgg tcccggccga ggccggcgca gggagggagg
agccgcccgg 60 gctgtggggg cgccgcgagc tgggccggcc tcggtgtgcc
cgcgccgcca gcccgctcca 120 gacgcgccac ctgggcgctc caagaagagg
ccgaagtttg ccgcggccgt gagttggagc 180 tcgcgccggg ccgctgcgcc
gggagctccg ggggcttccc tcgcttcccg gtattgtttg 240 caaactttgc
tgctctccgc cgcggccccc aactcggcgg acgccgggcg cggagagccg 300
agccgggggc gctgtgcgca gcgctcgggc caggccgggc gggcatgggc gggggcccga
360 gcaggggtgg agagccgggg ccagcagcag cccgtgcccg ggagcggcgg
cgctgagggg 420 cgcggagctc cccgcgagga cacgtccaac gccagcatgc
agcgcccggg cccccgcctg 480 tggctggtcc tgcaggtgat gggctcgtgc
gccgccatca gctccatgga catggagcgc 540 ccgggcgacg gcaaatgcca
gcccatcgag atcccgatgt gcaaggacat cggctacaac 600 atgactcgta
tgcccaacct gatgggccac gagaaccagc gcgaggcagc catccagttg 660
cacgagttcg cgccgctggt ggagtacggc tgccacggcc acctccgctt cttcctgtgc
720 tcgctgtacg cgccgatgtg caccgagcag gtctctaccc ccatccccgc
ctgccgggtc 780 atgtgcgagc aggcccggct caagtgctcc ccgattatgg
agcagttcaa cttcaagtgg 840 cccgactccc tggactgccg gaaactcccc
aacaagaacg accccaacta cctgtgcatg 900 gaggcgccca acaacggctc
ggacgagccc acccggggct cgggcctgtt cccgccgctg 960 ttccggccgc
agcggcccca cagcgcgcag gagcacccgc tgaaggacgg gggccccggg 1020
cgcggcggct gcgacaaccc gggcaagttc caccacgtgg agaagagcgc gtcgtgcgcg
1080 ccgctctgca cgcccggcgt ggacgtgtac tggagccgcg aggacaagcg
cttcgcagtg 1140 gtctggctgg ccatctgggc ggtgctgtgc ttcttctcca
gcgccttcac cgtgctcacc 1200 ttcctcatcg acccggcccg cttccgctac
cccgagcgcc ccatcatctt cctctccatg 1260 tgctactgcg tctactccgt
gggctacctc atccgcctct tcgccggcgc cgagagcatc 1320 gcctgcgacc
gggacagcgg ccagctctat gtcatccagg agggactgga gagcaccggc 1380
tgcacgctgg tcttcctggt cctctactac ttcggcatgg ccagctcgct gtggtgggtg
1440 gtcctcacgc tcacctggtt cctggccgcc ggcaagaagt ggggccacga
ggccatcgaa 1500 gccaacagca gctacttcca cctggcagcc tgggccatcc
cggcggtgaa gaccatcctg 1560 atcctggtca tgcgcagggt ggcgggggac
gagctcaccg gggtctgcta cgtgggcagc 1620 atggacgtca acgcgctcac
cggcttcgtg ctcattcccc tggcctgcta cctggtcatc 1680 ggcacgtcct
tcatcctctc gggcttcgtg gccctgttcc acatccggag ggtgatgaag 1740
acgggcggcg agaacacgga caagctggag aagctcatgg tgcgtatcgg gctcttctct
1800 gtgctgtaca ccgtgccggc cacctgtgtg atcgcctgct acttttacga
acgcctcaac 1860 atggattact ggaagatcct ggcggcgcag cacaagtgca
aaatgaacaa ccagactaaa 1920 acgctggact gcctgatggc cgcctccatc
cccgccgtgg agatcttcat ggtgaagatc 1980 tttatgctgc tggtggtggg
gatcaccagc gggatgtgga tttggacctc caagactctg 2040 cagtcctggc
agcaggtgtg cagccgtagg ttaaagaaga agagccggag aaaaccggcc 2100
agcgtgatca ccagcggtgg gatttacaaa aaagcccagc atccccagaa aactcaccac
2160 gggaaatatg agatccctgc ccagtcgccc acctgcgtgt gaacagggct
ggagggaagg 2220 gcacaggggc gcccggagct aagatgtggt gcttttcttg
gttgtgtttt tctttcttct 2280 tcttcttttt ttttttttat aaaagcaaaa
gagaaataca taaaaaagtg tttaccctga 2340 aattcaggat gctgtgatac
actgaaagga aaaatgtact taaagggttt tgttttgttt 2400 tggttttcca
gcgaagggaa gctcctccag tgaagtagcc tcttgtgtaa ctaatttgtg 2460
gtaaagtagt tgattcagcc ctcagaagaa aacttttgtt tagagccctc cctaaatata
2520 catctgtgta tttgagttgg ctttgctacc catttacaaa taagaggaca
gataactgct 2580 ttgcaaattc aagagcctcc cctgggttaa caaatgagcc
atccccaggg cccaccccca 2640 ggaaggccac agtgctgggc ggcatccctg
cagaggaaag acaggacccg gggcccgcct 2700 cacaccccag tggatttgga
gttgcttaaa atagactccg gccttcacca atagtctctc 2760 tgcaagacag
aaacctccat caaacctcac atttgtgaac tcaaacgatg tgcaatacat 2820
ttttttctct ttccttgaaa ataaaaagag aaacaagtat tttgctatat ataaagacaa
2880 caaaagaaat ctcctaacaa aagaactaag aggcccagcc ctcagaaacc
cttcagtgct 2940 acattttgtg gctttttaat ggaaaccaag ccaatgttat
agacgtttgg actgatttgt 3000 ggaaaggagg ggggaagagg gagaaggatc
attcaaaagt tacccaaagg gcttattgac 3060 tctttctatt gttaaacaaa
tgatttccac aaacagatca ggaagcacta ggttggcaga 3120 gacactttgt
ctagtgtatt ctcttcacag tgccaggaaa gagtggtttc tgcgtgtgta 3180
tatttgtaat atatgatatt tttcatgctc cactatttta ttaaaaataa aatatgttct
3240 ttagtttgct gctaaaaaaa 3260 59 49 PRT Artificial Sequence
Description of Artificial Sequenceportion of first extracellular
region of human frizzled1 (HFZ1) 59 Val Gly Gln Asn Thr Ser Asp Lys
Gly Thr Pro Ser Leu Leu Pro Glu 1 5 10 15 Phe Trp Thr Ser Asn Pro
Gln His Gly Gly Gly Gly His Arg Gly Gly 20 25 30 Phe Pro Gly Gly
Ala Gly Ala Ser Glu Arg Gly Lys Phe Ser Cys Pro 35 40 45 Arg 60 51
PRT Artificial Sequence Description of Artificial Sequenceportion
of first extracellular region of human frizzled2 (HFZ2) 60 Val Gly
Gln Asn His Ser Glu Asp Gly Ala Pro Ala Leu Leu Thr Thr 1 5 10 15
Ala Pro Pro Pro Gly Leu Gln Pro Gly Ala Gly Gly Thr Pro Gly Gly 20
25 30 Pro Gly Gly Gly Gly Ala Pro Pro Arg Tyr Ala Thr Leu Glu His
Pro 35 40 45 Phe His Cys 50 61 26 PRT Artificial Sequence
Description of Artificial Sequenceportion of first extracellular
region of human frizzled3 (HFZ3) 61 Leu Val Asp Leu Asn Leu Ala Gly
Glu Pro Thr Glu Gly Ala Pro Val 1 5 10 15 Ala Val Gln Arg Asp Tyr
Gly Phe Trp Cys 20 25 62 20 PRT Artificial Sequence Description of
Artificial Sequenceportion of first extracellular region of human
frizzled4 (HFZ4) 62 Cys Met Glu Gly Pro Gly Asp Glu Glu Val Pro Leu
Pro His Lys Thr 1 5 10 15 Pro Ile Gln Pro 20 63 46 PRT Artificial
Sequence Description of Artificial Sequenceportion of first
extracellular region of human frizzled5 (HFZ5) 63 Cys Met Asp Tyr
Asn Arg Ser Glu Ala Thr Thr Ala Pro Pro Arg Pro 1 5 10 15 Phe Pro
Ala Lys Pro Thr Leu Pro Gly Pro Pro Gly Ala Pro Ala Ser 20 25 30
Gly Gly Glu Cys Pro Ala Gly Gly Pro Phe Val Cys Lys Cys 35 40 45 64
26 PRT Artificial Sequence Description of Artificial
Sequenceportion of first extracellular region of human frizzled6
(HFZ6) 64 Thr Phe Asp Pro His Thr Glu Phe Leu Gly Pro Gln Lys Lys
Thr Glu 1 5 10 15 Gln Val Gln Arg Asp Ile Gly Phe Trp Cys 20 25 65
50 PRT Artificial Sequence Description of Artificial
Sequenceportion of first extracellular region of human frizzled7
(HFZ7) 65 Val Gly Gln Asn Thr Ser Asp Gly Ser Gly Gly Pro Gly Gly
Gly Pro 1 5 10 15 Thr Ala Tyr Pro Thr Ala Pro Tyr Leu Pro Asp Leu
Pro Phe Thr Ala 20 25 30 Leu Pro Pro Gly Ala Ser Asp Gly Arg Gly
Arg Pro Ala Phe Pro Phe 35 40 45 Ser Cys 50 66 87 PRT Artificial
Sequence Description of Artificial Sequenceportion of first
extracellular region of human frizzled8 (HFZ8) 66 Cys Met Asp Tyr
Asn Arg Thr Asp Leu Thr Thr Ala Ala Pro Ser Pro 1 5 10 15 Pro Arg
Arg Leu Pro Pro Pro Pro Pro Gly Glu Gln Pro Pro Ser Gly 20 25 30
Ser Gly His Gly Arg Pro Pro Gly Ala Arg Pro Pro His Arg Gly Gly 35
40 45 Gly Arg Gly Gly Gly Gly Gly Asp Ala Ala Ala Pro Pro Ala Arg
Gly 50 55 60 Gly Gly Gly Gly Gly Lys Ala Arg Pro Pro Gly Gly Gly
Ala Ala Pro 65 70 75 80 Cys Glu Pro Gly Cys Gln Cys 85 67 37 PRT
Artificial Sequence Description of Artificial Sequenceportion of
first extracellular region of human frizzled9 (HFZ9) 67 Cys Met Glu
Ala Pro Glu Asn Ala Thr Ala Gly Pro Ala Glu Pro His 1 5 10 15 Lys
Gly Leu Gly Met Leu Pro Val Ala Pro Arg Pro Ala Arg Pro Pro 20 25
30 Gly Asp Leu Gly Pro 35 68 38 PRT Artificial Sequence Description
of Artificial Sequenceportion of first extracellular region of
human frizzled10 (HFZ10) 68 Asn Tyr Leu Cys Met Glu Ala Pro Asn Asn
Gly Ser Asp Glu Pro Thr 1 5 10 15 Arg Gly Ser Gly Leu Phe Pro Pro
Leu Phe Arg Pro Gln Arg Pro His 20 25 30 Ser Ala Gln Glu His Pro 35
69 22 DNA Artificial Sequence Description of Artificial
Sequencereal-time PCR Wnt1 forward primer 69 cgaacctgct tacagactcc
aa 22 70 17 DNA Artificial Sequence Description of Artificial
Sequencereal-time PCR Wnt1 reverse primer 70 cagacgccgc tgtttgc 17
71 26 DNA Artificial Sequence Description of Artificial
SequenceWnt1 probe 71 tgcaactggt actcgagccc agtctg 26 72 23 DNA
Artificial Sequence Description of Artificial Sequencereal-time PCR
Wnt2 forward primer 72 ggatgaccaa gtgtgggtgt aag 23 73 20 DNA
Artificial Sequence Description of Artificial Sequencereal-time PCR
Wnt2 reverse primer 73 gtgcacatcc agagcttcca 20 74 19 DNA
Artificial Sequence Description of Artificial SequenceWnt2 probe 74
cactggtgct gcgccgtgc 19 75 23 DNA Artificial Sequence Description
of Artificial Sequencereal-time PCR Wnt2b forward primer 75
ggcacgagtg atctgtgaca ata 23 76 20 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wnt2b reverse
primer 76 cgcatgatgt ctgggtaacg 20 77 19 DNA Artificial Sequence
Description of Artificial SequenceWnt2b probe 77 tttggtgagc
cggcagcgg 19 78 21 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR Wnt3 forward primer 78 ctgggccagc
agtacacatc t 21 79 22 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR Wnt3 reverse primer 79 ggcatgatct
cgatgtaatt gc 22 80 20 DNA Artificial Sequence Description of
Artificial SequenceWnt3 probe 80 tgctctgcgg ctccatccca
20 81 18 DNA Artificial Sequence Description of Artificial
Sequencereal-time PCR Wnt3a forward primer 81 cccgtgctgg acaaagct
18 82 21 DNA Artificial Sequence Description of Artificial
Sequencereal-time PCR Wnt3a reverse primer 82 tctgcacatg agcgtgtcac
t 21 83 22 DNA Artificial Sequence Description of Artificial
SequenceWnt3a probe 83 ttgtccacgc cattgcctca gc 22 84 20 DNA
Artificial Sequence Description of Artificial Sequencereal-time PCR
Wnt4 forward primer 84 ggaggagacg tgcgagaaac 20 85 20 DNA
Artificial Sequence Description of Artificial Sequencereal-time PCR
Wnt4 reverse primer 85 caggttccgc ttgcacatct 20 86 23 DNA
Artificial Sequence Description of Artificial SequenceWnt4 probe 86
caagggcctg atccagaggc agg 23 87 20 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wnt5a forward
primer 87 tctccttcgc ccaggttgta 20 88 26 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wnt5a reverse
primer 88 cttctgacat ctgaacaggg ttattc 26 89 27 DNA Artificial
Sequence Description of Artificial SequenceWnt5a probe 89
tgaagccaat tcttggtggt cgctagg 27 90 22 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wnt5b forward
primer 90 ccaactcctg gtggtcatta gc 22 91 21 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wnt5b reverse
primer 91 tgggcaccga tgataaacat c 21 92 22 DNA Artificial Sequence
Description of Artificial SequenceWnt5b probe 92 ttgaacccgg
tgcagagacc cg 22 93 16 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR Wnt6 forward primer 93 tccgccgctg
gaattg 16 94 18 DNA Artificial Sequence Description of Artificial
Sequencereal-time PCR Wnt6 reverse primer 94 aggccgtctc ccgaatgt 18
95 22 DNA Artificial Sequence Description of Artificial
SequenceWnt6 probe 95 aggcctttgg acgcatcctg ca 22 96 21 DNA
Artificial Sequence Description of Artificial Sequencereal-time PCR
Wnt7a forward primer 96 gacgccatca tcgtcatagg a 21 97 18 DNA
Artificial Sequence Description of Artificial Sequencereal-time PCR
Wnt7a reverse primer 97 ggccattgcg gaactgaa 18 98 25 DNA Artificial
Sequence Description of Artificial SequenceWnt7a probe 98
tcacaaatgg gcctggacga gtgtc 25 99 20 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wnt7b forward
primer 99 tgaagctcgg agcactgtca 20 100 19 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wnt7b reverse
primer 100 ggccaggaat cttgttgca 19 101 20 DNA Artificial Sequence
Description of Artificial SequenceWnt7b probe 101 tggtggccct
gggagccaac 20 102 20 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR Wnt8a forward primer 102
gcagaggcgg aactgatctt 20 103 21 DNA Artificial Sequence Description
of Artificial Sequencereal-time PCR Wnt8a reverse primer 103
cgaccctctg tgccatagat g 21 104 30 DNA Artificial Sequence
Description of Artificial SequenceWnt8a probe 104 ccagattact
gtacctgcaa ttccagcctg 30 105 21 DNA Artificial Sequence Description
of Artificial Sequencereal-time PCR Wnt8b forward primer 105
aatcgggaga cagcatttgt g 21 106 24 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wnt8b reverse
primer 106 atctccaagg ctgcagtttc tagt 24 107 29 DNA Artificial
Sequence Description of Artificial SequenceWnt8b probe 107
tgccatcagt tctgctggag tcatgtaca 29 108 22 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wnt10a forward
primer 108 ctgggtgctc ctgttcttcc ta 22 109 19 DNA Artificial
Sequence Description of Artificial Sequencereal-time PCR Wnt10a
reverse primer 109 gaggcggagg tccagaatg 19 110 22 DNA Artificial
Sequence Description of Artificial SequenceWnt10a probe 110
ctgccatgcc caggtcagca cc 22 111 18 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wnt10b forward
primer 111 cctcgcgggt ctcctgtt 18 112 22 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wnt10b reverse
primer 112 aggcccagaa tctcattgct ta 22 113 21 DNA Artificial
Sequence Description of Artificial SequenceWnt10b probe 113
ctggcgttgt gcagtcgggc t 21 114 21 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wnt11 forward
primer 114 cgtgtgctat ggcatcaagt g 21 115 20 DNA Artificial
Sequence Description of Artificial Sequencereal-time PCR Wnt11
reverse primer 115 gcagtgttgc gtctggttca 20 116 23 DNA Artificial
Sequence Description of Artificial SequenceWnt11 probe 116
tgtccaagac accatcggcc ctg 23 117 18 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wnt14 forward
primer 117 gggcagacgg tcaagcaa 18 118 21 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wnt14 reverse
primer 118 ccagccttga tcaccttcac a 21 119 22 DNA Artificial
Sequence Description of Artificial SequenceWnt14 probe 119
ctgcgagccc gtgtggactt cc 22 120 18 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wnt16 forward
primer 120 gccaatttgc cgctgaac 18 121 18 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wnt16 reverse
primer 121 cggcagcagg tacggttt 18 122 23 DNA Artificial Sequence
Description of Artificial SequenceWnt16 probe 122 ccgccagaag
gagctgtgca aga 23 123 19 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR Fzd1 forward primer 123 caccttgtga
gccgaccaa 19 124 21 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR Fzd1 reverse primer 124 cagcactgac
caaatgccaa t 21 125 20 DNA Artificial Sequence Description of
Artificial SequenceFzd1 probe 125 aggagctgcg cttctcgcgc 20 126 18
DNA Artificial Sequence Description of Artificial Sequencereal-time
PCR Fzd2 forward primer 126 tttctgggcg agcgtgat 18 127 20 DNA
Artificial Sequence Description of Artificial Sequencereal-time PCR
Fzd2 reverse primer 127 aaacgcgtct cctcctgtga 20 128 17 DNA
Artificial Sequence Description of Artificial SequenceFzd2 probe
128 tgcgaacctg cgcggcc 17 129 22 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Fzd3 forward primer
129 tggctatggt ggatgatcaa ag 22 130 17 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Fzd3 reverse primer
130 tggaggctgc cgtggta 17 131 27 DNA Artificial Sequence
Description of Artificial SequenceFzd3 probe 131 aggaagcatc
cacagcaaag tgagcag 27 132 20 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR Fzd4 forward primer 132 ggcggcatgt
gtctttcagt 20 133 25 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR Fzd4 reverse primer 133 gaatttgctg
cagttcagac tctct 25 134 26 DNA Artificial Sequence Description of
Artificial SequenceFzd4 probe 134 agagacgctg tgaacccgtc ctgaag 26
135 18 DNA Artificial Sequence Description of Artificial
Sequencereal-time PCR Fzd5 forward primer 135 cgcgagcaca accacatc
18 136 25 DNA Artificial Sequence Description of Artificial
Sequencereal-time PCR Fzd5 reverse primer 136 agaagtagac caggaggaag
acgat 25 137 22 DNA Artificial Sequence Description of Artificial
SequenceFzd5 probe 137 tacgagacca cgggccctgc ac 22 138 23 DNA
Artificial Sequence Description of Artificial Sequencereal-time PCR
Fzd6 forward primer 138 acaagctgaa ggtcatttcc aaa 23 139 22 DNA
Artificial Sequence Description of Artificial Sequencereal-time PCR
Fzd6 reverse primer 139 gctactgcag aagtgccatg at 22 140 26 DNA
Artificial Sequence Description of Artificial SequenceFzd6 probe
140 atgggaacca gcacaggagc tacagc 26 141 23 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Fzd7 forward primer
141 caacggcctg atgtacttta agg 23 142 22 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Fzd7 reverse primer
142 catgtccacc aggtaggtga ga 22 143 23 DNA Artificial Sequence
Description of Artificial SequenceFzd7 probe 143 ctgcgcctcg
acgctcttta ccg 23 144 20 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR Fzd8 forward primer 144 gctcggtcat
caagcaacag 20 145 21 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR Fzd8 reverse primer 145 acggtgtaga
gcacggtgaa c 21 146 22 DNA Artificial Sequence Description of
Artificial SequenceFzd8 probe 146 aagctgatga tccgcctggg cc 22 147
20 DNA Artificial Sequence Description of Artificial
Sequencereal-time PCR Fzd9 forward primer 147 gcgctcaaga ccatcgtcat
20 148 18 DNA Artificial Sequence Description of Artificial
Sequencereal-time PCR Fzd9 reverse primer 148 149 22 DNA Artificial
Sequence Description of Artificial SequenceFzd9 probe 149
tggcgggtga tgagctgact gg 22 150 17 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Fzd10 forward
primer 150 gccgccatca gctccat 17 151 22 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Fzd10 reverse
primer 151 tcatgttgta gccgatgtcc tt 22 152 22 DNA Artificial
Sequence Description of Artificial SequenceFzd10 probe 152
atgccagccc atcgagatcc cg 22 153 23 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Frp1 forward primer
153 agcgagtacg actacgtgag ctt 23 154 21 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Frp1 reverse primer
154 gcactgaggt ggcttggtgt a 21 155 23 DNA Artificial Sequence
Description of Artificial SequenceFrp1 probe 155 agtcggacat
cggcccgtac cag 23 156 24 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR Frp2 forward primer 156 agaccaagag
caagaccatt taca 24 157 19 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR Frp2 reverse primer 157 ttgagccaca
gcaccgatt 19 158 23 DNA Artificial Sequence Description of
Artificial SequenceFrp2 probe 158 cggtgtgtcc gaaagggacc tga 23 159
22 DNA Artificial Sequence Description of Artificial
Sequencereal-time PCR Frp3 forward primer 159 gggctatgaa gatgaggaac
gt 22 160 21 DNA Artificial Sequence Description of Artificial
Sequencereal-time PCR Frp3 reverse primer 160 ccgagtcgat ccttccactt
c 21 161 34 DNA Artificial Sequence Description of Artificial
SequenceFrp3 probe 161 ccagattact cttggtggaa ggctctatag ctga 34 162
24 DNA Artificial Sequence Description of Artificial
Sequencereal-time PCR Frp4 forward primer 162 cggaggatgt taagtggata
gaca 24 163 24 DNA Artificial Sequence Description of Artificial
Sequencereal-time PCR Frp4 reverse primer 163 aggcgtttac agtcaacatc
aaga 24 164 29 DNA Artificial Sequence Description of Artificial
SequenceFrp4 probe 164 cacaccagac atgatggtac aggaaaggc 29 165 20
DNA Artificial Sequence Description of Artificial Sequencereal-time
PCR Frp5 forward primer 165 agctgattgg agcccagaaa 20 166 18 DNA
Artificial Sequence Description of Artificial Sequencereal-time PCR
Frp5 reverse primer 166 tggtgtcctt gcgcttca 18 167 22 DNA
Artificial Sequence Description of Artificial SequenceFrp5 probe
167 aagaagctgc tcaagccggg cc 22 168 19 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wisp1 forward
primer 168 cctgatgggc ttggcttct 19 169 22 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wisp1 reverse
primer 169 tgggattcct acagctcagg tt 22 170 26 DNA Artificial
Sequence Description of Artificial SequenceWisp1 probe 170
ccgccaggtc ctatggatta atgcct 26 171 19 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wisp2 forward
primer 171 acccacctcc tggccttct 19 172 18 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Wisp2 reverse
primer 172 agcagccaca gccatcca 18 173 28 DNA Artificial Sequence
Description of Artificial SequenceWisp2 probe 173 tcctctgcct
cctctcaaag gtgcgtac 28 174 20 DNA Artificial Sequence Description
of Artificial Sequencereal-time PCR Wisp3 forward primer 174
aaagctggct ggcagtcact 20 175 23 DNA Artificial Sequence Description
of Artificial Sequencereal-time PCR Wisp3 reverse primer 175
aatggttcca ggctacagtt tga 23 176 32 DNA Artificial Sequence
Description of Artificial SequenceWisp3 probe 176 tctggagcta
aaggtggaaa gaagtctgat ca 32 177 26 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR DKK1 forward primer
177 ggaataagta ccagaccatt gacaac 26 178 22 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR DKK1 reverse primer
178 gggactagcg cagtactcat ca 22 179 20 DNA Artificial Sequence
Description of Artificial SequenceDKK1 probe 179 cagccgtacc
cgtgcgcaga 20 180 22 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR DKK2 forward primer 180 ctgatggtgg
agagctcaca ga 22 181 25 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR DKK2 reverse primer 181 cagagaggac
ttgatggagt tgagt 25 182 17 DNA Artificial Sequence Description of
Artificial SequenceDKK2 probe 182
cggcagttcg cgggcca 17 183 19 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR DKK3 forward primer 183 ggaggacacg
cagcacaaa 19 184 24 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR DKK3 reverse primer 184 caggttcact
tctgatgatg cttt 24 185 18 DNA Artificial Sequence Description of
Artificial SequenceDKK3 probe 185 tgcgcagcgc ggtggaag 18 186 23 DNA
Artificial Sequence Description of Artificial Sequencereal-time PCR
DKK4 forward primer 186 ggcataaaga cactgctcaa gct 23 187 19 DNA
Artificial Sequence Description of Artificial Sequencereal-time PCR
DKK4 reverse primer 187 gctggtcaat tggcttcga 19 188 22 DNA
Artificial Sequence Description of Artificial SequenceDKK4 probe
188 cgttgcgact gtggccctgg ac 22 189 20 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR IL-6 forward primer
189 cctgacccaa ccacaaatgc 20 190 22 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR IL-6 reverse primer
190 gcgcagaatg agatgagttg tc 22 191 25 DNA Artificial Sequence
Description of Artificial SequenceIL-6 probe 191 ctgacgaagc
tgcaggcaca gaacc 25 192 20 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR C-Myc forward primer 192
gccacgtctc cacacatcag 20 193 22 DNA Artificial Sequence Description
of Artificial Sequencereal-time PCR C-Myc reverse primer 193
tcttggcagc aggatagtcc tt 22 194 20 DNA Artificial Sequence
Description of Artificial SequenceC-Myc probe 194 cgcagcgcct
ccctccactc 20 195 22 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR Fibro forward primer 195
cacccaattc cttgctggta tc 22 196 23 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Fibro reverse
primer 196 cccaggcttc tcatacttga tga 23 197 22 DNA Artificial
Sequence Description of Artificial SequenceFibro probe 197
agccgccacg tgccaggatt ac 22 198 20 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Cyc D1 forward
primer 198 ggcggaggag aacaaacaga 20 199 18 DNA Artificial Sequence
Description of Artificial Sequencereal-time PCR Cyc D1 reverse
primer 199 tggcacaaga ggcaacga 18 200 20 DNA Artificial Sequence
Description of Artificial SequenceCyc D1 probe 200 tccgcaaaca
cgcgcagacc 20 201 23 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR MMP3 forward primer 201 ccatcagagg
aaatgaggta cga 23 202 19 DNA Artificial Sequence Description of
Artificial Sequencereal-time PCR MMP3 reverse primer 202 cctcacggtt
ggagggaaa 19 203 26 DNA Artificial Sequence Description of
Artificial SequenceMMP3 probe 203 ctggataccc aagaggcatc cacacc 26
204 19 DNA Artificial Sequence Description of Artificial
Sequencereal-time PCR LRP5 forward primer 204 cgtgattgcc gacgatctc
19 205 19 DNA Artificial Sequence Description of Artificial
Sequencereal-time PCR LRP5 reverse primer 205 tccggccgct agtcttgtc
19 206 25 DNA Artificial Sequence Description of Artificial
SequenceLRP5 probe 206 acccgttcgg tctgacgcag tacag 25 207 5 PRT
Artificial Sequence Description of Artificial Sequencepeptide
linker 207 Gly Gly Gly Gly Ser 1 5 208 24 DNA Artificial Sequence
Description of Artificial SequenceFzd-2 PCR amplification reverse
primer 208 cagcgtcttg cccgaccaga tcca 24 209 24 DNA Artificial
Sequence Description of Artificial SequenceFzd-2 PCR amplification
forward primer 209 ctagcgccgc tcttcgtgta cctg 24 210 21 DNA
Artificial Sequence Description of Artificial SequenceFzd-5 PCR
amplification forward primer 210 ttcatgtgcc tggtggtggg c 21 211 21
DNA Artificial Sequence Description of Artificial SequenceFzd-5 PCR
amplification reverse primer 211 tacacgtgcg acagggacac c 21 212 21
DNA Artificial Sequence Description of Artificial SequenceWnt-1 PCR
amplification forward primer 212 cacgacctcg tctacttcga c 21 213 21
DNA Artificial Sequence Description of Artificial SequenceWnt-1 PCR
amplification reverse primer 213 acagacactc gtgcagtacg c 21 214 21
DNA Artificial Sequence Description of Artificial SequenceWnt-5a
PCR amplification forward primer 214 acacctcttt ccaaacaggc c 21 215
21 DNA Artificial Sequence Description of Artificial SequenceWnt-5a
PCR amplification reverse primer 215 ggattgttaa actcaactct c 21 216
21 DNA Artificial Sequence Description of Artificial SequenceWnt-7a
PCR amplification forward primer 216 cgcaacaagc ggcccacctt c 21 217
21 DNA Artificial Sequence Description of Artificial SequenceWnt-7a
PCR amplification reverse primer 217 tccgtgcgct cgctgcacgt g 21 218
22 DNA Artificial Sequence Description of Artificial
SequenceWnt-10b PCR amplification forward primer 218 gaatgcgaat
ccacaacaac ag 22 219 23 DNA Artificial Sequence Description of
Artificial SequenceWnt-10b PCR amplification reverse primer 219
ttgcggttgt gggtatcaat gaa 23 220 21 DNA Artificial Sequence
Description of Artificial SequenceWnt-13 PCR amplification forward
primer 220 aagatggtgc caacttcacc g 21 221 21 DNA Artificial
Sequence Description of Artificial SequenceWnt-13 PCR amplification
reverse primer 221 ctgccttctt gggggctttg c 21 222 20 DNA Artificial
Sequence Description of Artificial SequenceG3PDH PCR amplification
forward primer 222 accacagtcc atgccatcac 20 223 20 DNA Artificial
Sequence Description of Artificial SequenceG3PDH PCR amplification
reverse primer 223 tacagcaaca gggtggtgga 20 224 4 PRT Artificial
Sequence Description of Artificial Sequenceshort linker sequence
224 Gly Pro Ser Leu 1 225 75 PRT Artificial Sequence Description of
Artificial SequencepFZD2-TT tetanus toxin epitope fused to frizzled
domain 225 Met Cys Val Gly Gln Asn His Ser Glu Asp Gly Ala Pro Ala
Leu Leu 1 5 10 15 Thr Thr Ala Pro Pro Pro Gly Leu Gln Pro Gly Ala
Gly Gly Thr Pro 20 25 30 Gly Gly Pro Gly Gly Gly Gly Ala Pro Pro
Arg Tyr Ala Thr Leu Glu 35 40 45 His Pro Phe His Cys Gly Pro Ser
Leu Val Asp Asp Ala Leu Ile Asn 50 55 60 Ser Thr Lys Ile Tyr Ser
Tyr Phe Pro Ser Val 65 70 75 226 228 DNA Artificial Sequence
Description of Artificial SequencepFZD2-TT tetanus toxin epitope
fused to frizzled domain 226 atgtgcgtcg gccagaacca ctccgaggac
ggagctcccg cgctactcac caccgcgccg 60 ccgccgggac tgcagccggg
tgccgggggc accccgggtg gcccgggcgg cggcggcgct 120 cccccgcgct
acgccacgct ggagcacccc ttccactgcg gccccagcct ggtggacgac 180
gccctgatca acagcaccaa gatctacagc tactttccca gcgtgtag 228 227 75 PRT
Artificial Sequence Description of Artificial SequencepTT-FZD2
tetanus toxin epitope fused to frizzled domain 227 Met Val Asp Asp
Ala Leu Ile Asn Ser Thr Lys Ile Tyr Ser Tyr Phe 1 5 10 15 Pro Ser
Val Gly Pro Ser Leu Cys Val Gly Gln Asn His Ser Glu Asp 20 25 30
Gly Ala Pro Ala Leu Leu Thr Thr Ala Pro Pro Pro Gly Leu Gln Pro 35
40 45 Gly Ala Gly Gly Thr Pro Gly Gly Pro Gly Gly Gly Gly Ala Pro
Pro 50 55 60 Arg Tyr Ala Thr Leu Glu His Pro Phe His Cys 65 70 75
228 228 DNA Artificial Sequence Description of Artificial
SequencepTT-FZD2 tetanus toxin epitope fused to frizzled domain 228
atggtggacg acgccctgat caacagcacc aagatctaca gctactttcc cagcgtgggc
60 cccagcctgt gcgtcggcca gaaccactcc gaggacggag ctcccgcgct
actcaccacc 120 gcgccgccgc cgggactgca gccgggtgcc gggggcaccc
cgggtggccc gggcggcggc 180 ggcgctcccc cgcgctacgc cacgctggag
caccccttcc actgctag 228 229 75 PRT Artificial Sequence Description
of Artificial SequencePFZD2-MMVF measles virus fusion (MVF) epitope
fused to frizzled domain 229 Met Cys Val Gly Gln Asn His Ser Glu
Asp Gly Ala Pro Ala Leu Leu 1 5 10 15 Thr Thr Ala Pro Pro Pro Gly
Leu Gln Pro Gly Ala Gly Gly Thr Pro 20 25 30 Gly Gly Pro Gly Gly
Gly Gly Ala Pro Pro Arg Tyr Ala Thr Leu Glu 35 40 45 His Pro Phe
His Cys Gly Pro Ser Leu Lys Leu Leu Ser Leu Ile Lys 50 55 60 Gly
Val Ile Val His Arg Leu Glu Gly Val Glu 65 70 75 230 228 DNA
Artificial Sequence Description of Artificial SequencePFZD2-MMVF
measles virus fusion (MVF) epitope fused to frizzled domain 230
atgtgcgtcg gccagaacca ctccgaggac ggagctcccg cgctactcac caccgcgccg
60 ccgccgggac tgcagccggg tgccgggggc accccgggtg gcccgggcgg
cggcggcgct 120 cccccgcgct acgccacgct ggagcacccc ttccactgcg
gccccagcct gaagctgctg 180 agcctgatca agggcgtgat cgtgcaccgc
ctggagggcg tggagtag 228 231 75 PRT Artificial Sequence Description
of Artificial SequencePMMVF-ZD2 measles virus fusion (MVF) epitope
fused to frizzled domain 231 Met Lys Leu Leu Ser Leu Ile Lys Gly
Val Ile Val His Arg Leu Glu 1 5 10 15 Gly Val Glu Gly Pro Ser Leu
Cys Val Gly Gln Asn His Ser Glu Asp 20 25 30 Gly Ala Pro Ala Leu
Leu Thr Thr Ala Pro Pro Pro Gly Leu Gln Pro 35 40 45 Gly Ala Gly
Gly Thr Pro Gly Gly Pro Gly Gly Gly Gly Ala Pro Pro 50 55 60 Arg
Tyr Ala Thr Leu Glu His Pro Phe His Cys 65 70 75 232 228 DNA
Artificial Sequence Description of Artificial SequencePMMVF-ZD2
measles virus fusion (MVF) epitope fused to frizzled domain 232
atgaagctgc tgagcctgat caagggcgtg atcgtgcacc gcctggaggg cgtggagggc
60 cccagcctgt gcgtcggcca gaaccactcc gaggacggag ctcccgcgct
actcaccacc 120 gcgccgccgc cgggactgca gccgggtgcc gggggcaccc
cgggtggccc gggcggcggc 180 ggcgctcccc cgcgctacgc cacgctggag
caccccttcc actgctag 228
* * * * *
References